Series Editors Leslie Wilson Department of Molecular, Cellular and Developmental Biology University of California Santa Barbara, California
Paul Matsudaira Department of Biological Sciences National University of Singapore Singapore
Academic Press is an imprint of Elsevier 30 Corporate Drive, Suite 400, Burlington, MA 01803, USA 525 B Street, Suite 1900, San Diego, CA 92101-4495, USA 32, Jamestown Road, London NW1 7BY, UK Linacre House, Jordan Hill, Oxford OX2 8DP, UK First edition 2010 Copyright ß 2010 Elsevier Inc. All rights reserved No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means electronic, mechanical, photocopying, recording or otherwise without the prior written permission of the publisher Permissions may be sought directly from Elsevier’s Science & Technology Rights Department in Oxford, UK: phone (+44) (0) 1865 843830; fax (+44) (0) 1865 853333; email:
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Printed and bound in USA 10 11 12 10 9 8 7 6 5 4 3 2 1
CONTRIBUTORS
Numbers in parentheses indicate the pages on which the authors’ contributions begin.
David L. Armstrong (183), Membrane Signaling Group, Laboratory of Neurobiology, National Institute of Environmental Health Sciences, NIH, Durham, North Carolina, USA Darryl A. Auston (113), Center for Biomedical Engineering and Technology, and Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland, USA S. Baudet (67), Ricerca Biosciences SAS, Saint Germain sur l’Arbresle, France D.M. Bers (67), Department of Pharmacology, University of California, Davis, Davis, California, USA Donald M. Bers (1), Department of Pharmacology, University of California, Davis School of Medicine, Davis, California, USA Francis Burton (225), School of Life Sciences, University of Glasgow, United Kingdom Christian Erxleben (183), Membrane Signaling Group, Laboratory of Neurobiology, National Institute of Environmental Health Sciences, NIH, Durham, North Carolina, USA L. Hove-Madsen (67), Cardiovascular Research Centre CSIC-ICCC, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain Joseph P.Y. Kao (113), Center for Biomedical Engineering and Technology, and Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland, USA Eric Karplus (263), Science Wares Inc., Falmouth, Massachusetts, USA Ole Johan Kemi (225), School of Life Sciences, University of Glasgow, United Kingdom Gong Li (113), Center for Biomedical Engineering and Technology, and Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland, USA Mark A. Messerli (91), BioCurrents Research Center, Cellular Dynamics Program, Marine Biological Laboratory, Woods Hole, Massachusetts, USA Andrew L. Miller (263), Biochemistry and Cell Biology Section and State Key Laboratory of Molecular Neuroscience, Division of Life Science, HKUST, Clear Water Bay, Kowloon, Hong Kong, PR China Richard Nuccitelli (1), BioElectroMed Corp., Burlingame, California, USA Chris W. Patton (1), Hopkins Marine Station, Stanford University, Pacific Grove California, USA Taufiq Rahman (199), Department of Pharmacology, Tennis Court Road, University of Cambridge, Cambridge, United Kingdom ix
x
Contributors
Martyn Reynolds (225), Cairn Research Limited, Faversham, Kent, United Kingdom Kelly L. Rogers (263), The Walter and Eliza Hall Institute of Medical Research, Parkville, Australia Godfrey Smith (225), School of Life Sciences, University of Glasgow, United Kingdom Peter J. S. Smith (91), BioCurrents Research Center, Cellular Dynamics Program, Marine Biological Laboratory, Woods Hole, Massachusetts, USA Colin W. Taylor (199), Department of Pharmacology, Tennis Court Road, University of Cambridge, Cambridge, United Kingdom Sarah E. Webb (263), Biochemistry and Cell Biology Section and State Key Laboratory of Molecular Neuroscience, Division of Life Science, HKUST, Clear Water Bay, Kowloon, Hong Kong, PR China Michael Whitaker (153), Institute of Cell and Molecular Biosciences, Medical School, Newcastle University, Framlington Place, Newcastle upon Tyne, United Kingdom Jody A. White (183), Membrane Signaling Group, Laboratory of Neurobiology, National Institute of Environmental Health Sciences, NIH, Durham, North Carolina, USA Robert Zucker (27), Molecular and Cell Biology Department, University of California at Berkeley, Berkeley, California, USA
PREFACE
This volume of Methods in Cell Biology is a sequel to the oft-consulted Volume 40 of the series edited by Richard Nuccitelli that oVered a practical guide to the study of calcium in living cells. Much in that volume remains relevant and this volume oVers updates of chapters contributed to the original volume. But in the decade and a half that have elapsed since the publication of Volume 40, as calcium signaling has continued to find itself a ubiquitous element of cell regulation, new technical advances have oVered themselves to the field and existing methods have been refined. This volume retains the bedrock of an understanding of calcium buVering and the manipulation of intracellular free calcium concentration in cells: the subtleties and peculiarities of an ion that acts at submicromolar concentrations and that is very actively regulated by cellular buVers and pumps are covered extensively by the early chapters on calcium buVers; a detailed treatment of dynamic changes in free calcium achieved by the photosensitive release of calcium from buVers that undergo light-induced changes in calcium aYnity follows on. Calcium-sensitive electrodes oVer the most quantitative approach to measuring calcium concentrations within cells and in solutions. Two chapters in this volume provide a deep understanding of both spatially homogeneous calcium sensing and of the use of calcium-sensitive electrodes to measure standing fluxes and gradients of calcium. Calcium-sensitive fluorescent dyes present some advantages in measuring intracellular free calcium over electrodes—what is lost in precision can be gained in convenience and time resolution. The two chapters on calcium-sensitive fluorescent approaches cover low molecular mass indicators and the newer recombinant techniques based on green fluorescent protein. In many circumstances, particularly in studying neuronal calcium signaling in individual neurones, patch clamp methods are king. Two chapters are devoted to patch clamp analysis of calcium signaling. One of these concentrates on calcium channels at the plasma membrane—an approach that remains key to understanding neuronal signaling mechanisms; the other highlights the remarkable achievement of using patch clamp techniques to study both the aggregate and single channel properties of calcium release channels in the membranes of intracellular calcium stores. The final two chapters of the volume explain the state-of-the-art in imaging calcium signals. Confocal and multiphoton microscopy have much improved the spatial and temporal resolution of the measurement of calcium signals, revealing, among other things, how very localized calcium signals play a part in the versatile xi
xii
Preface
repertoire of this key second messenger. Low-intensity photon imaging using aequorin has provided an approach best suited to the long-term recording of calcium signals associated with cell division and pattern formation, situations in which photobleaching and light-induced damage preclude the use of fluorescent probes. I thank all the authors of this volume for having made possible, as I see it, such a valuable and detailed contribution to the methodological state-of-the-art in the field. I also thank Zoe Kruze and Narmada Thangavelu of Elsevier for their help and patience in bringing this volume into being. Michael Whitaker
CHAPTER 1
A Practical Guide to the Preparation of Ca2þ BuVers Donald M. Bers,* Chris W. Patton,† and Richard Nuccitelli‡ *Department of Pharmacology University of California, Davis School of Medicine Davis, California, USA †
Hopkins Marine Station Stanford University, Pacific Grove California, USA ‡
BioElectroMed Corp. Burlingame, California, USA
Abstract I. Introduction II. Rationale A. Which Ca2þ BuVer Should You Use? B. EGTA: The Workhorse of Biological Ca2þ Chelators C. BAPTA Family of Ca2þ BuVers III. Methods A. Basic Mathematical Relationships B. Temperature, Ionic Strength, and pH Corrections IV. Materials A. [Ca2þ] Measurement and Calibration Solutions B. Preparing BuVer Solution C. Software Programs V. Discussion and Summary References
Abstract Calcium (Ca2þ) is a critical regulator of an immense array of biological processes, and the intracellular [Ca2þ] that regulates these processes is 10,000 lower than the extracellular [Ca2þ]. To study and understand these myriad Ca2þdependent functions requires control and measurement of [Ca2þ] in the nano- to micromolar range (where contaminating Ca2þ is a significant problem). As with METHODS IN CELL BIOLOGY, VOL. 99 Copyright 2010, Elsevier Inc. All rights reserved.
1
0091-679X/10 $35.00 DOI: 10.1016/S0091-679X(10)99001-8
Donald M. Bers et al.
2
pH, it is often essential to use Ca2þ buVers to control free [Ca2þ] at the desired biologically relevant concentrations. Fortunately, there are numerous available Ca2þ buVers with diVerent aYnities that make this practical. However, there are numerous caveats with respect to making these solutions appropriately with known Ca2þ buVers. These include pH dependence, selectivity for Ca2þ (e.g., vs. Mg2þ), ionic strength and temperature dependence, and complex multiple equilibria that occur in physiologically relevant solutions. Here we discuss some basic principles of Ca2þ buVering with respect to some of these caveats and provide practical tools (including freely downloadable computer programs) to help in the making and calibration of Ca2þ-buVered solutions for a wide array of biological applications.
I. Introduction Cell biologists quickly learn how important it is to control the ionic composition of the solutions used when studying cellular biochemistry, physiology, and pharmacology. BuVering the pH of the solutions we use is so routine that one can hardly imagine making a biological solution without the careful selection of the appropriate pH buVer and measurement of pH in the resulting solution. Indeed, there are an array of popular zwitterionic amino acid pH buVers introduced by Good et al. (1966) that are in widespread use (e.g., HEPES) and which complement the natural physiological pH buVers for these purposes. In contrast, there has been less attention to buVering and measuring [Ca2þ] because extracellular [Ca2þ] levels are typically in the millimolar range and such concentrations are easily measured and prepared. However, intracellular [Ca2þ] ([Ca2þ]i) is quite another matter because these levels are more typically in the 100 nM–10 mM range which is not as easily prepared or measured. For example, your source of distilled water could easily have trace Ca2þ contamination in the range of 1–10 mM. This range of contaminant Ca2þ can also come from chemicals and biochemicals commonly used to make solutions. Additionally, there is often a considerable amount of endogenous Ca2þ in biological tissue or cell samples which is not easily removed or controlled. Therefore, when we are interested in studying intracellular reactions, Ca2þ buVering is extremely important. In this chapter, we will present a practical guide to the preparation of Ca2þ buVer solutions. Our goal is to emphasize the methods and important variables to consider while making the procedure as simple as possible. We will also introduce computer programs which may be of practical use to many workers in this field. One is a spreadsheet useful in making and validating simple Ca2þ calibration solutions. The others are more powerful and extensive programs for the calculation of [Ca2þ] (and other metals and chelators) in complex solutions with multiple equilibria. These programs have been developed and described with maximum ease of use in mind.
1. A Practical Guide to the Preparation of Ca2þ BuVers
3
II. Rationale A. Which Ca2þ BuVer Should You Use? When selecting the appropriate Ca2þ buVer for your application, the main consideration is to choose one with a dissociation constant (Kd) close to the desired free [Ca2þ]. The ability of a buVer to absorb or release ions and thus to hold the solution at a given concentration of that ion is greatest at its Kd. Just as you should not choose PIPES (pKa ¼ 6.8) to buVer a solution at pH 7.8, choosing a Ca2þ buVer with a Kd far from the desired [Ca2þ] set point is a mistake. As a rule of thumb, the buVer’s Kd should not lie more than a factor of 10 from your desired [Ca2þ]. In addition, the buVer should exhibit a much greater aYnity for Ca2þ than Mg2þ since intracellular [Mg2þ] is typically 10,000-fold higher than [Ca2þ]i. Fortunately, about a dozen suitable buVers are available spanning the range from 10 nM to 100 mM (Table I). There are also a large number of fluorescent Ca2þ indicators (see Chapter 5) that can also serve as Ca2þ buVers, giving one the opportunity to both buVer and measure free [Ca2þ] with the same reagent. We will not focus on
Table I Mixed stability constants for useful Ca2þ buVers at 0.15 M ionic strength in order of Ca2þ aYnity log K0 Ca Ca2þ buVera
Kd
(pH 7.4)
K0 Ca (pH 7.4)/ K0 Ca (pH 7.0)
K0 Ca / K0 Mg (pH 7.4)
CDTA
7.90
13 nM
2.7
120
EGTA
7.18
67 nM
6.2
72,202
Quin 2 BAPTA Fura-2 Dibromo-BAPTA 4,40 -Difluoro-BAPTA Nitr-5 photolyzed 5-Methyl-50 -nitro-BAPTA 5-Mononitro-BAPTA NTA
6.84 6.71 6.61 5.74 5.77 5.2 4.66 4.4 3.87
144 nM 192 nM 242 nM 1.83 mM 1.7 mMb 6.3 mMb 22 mMb 40 mMb 134 mM
1.15 1.14 1.14 1.02 – – – – 2.5
25,114 158,244 72,373 63,000 – – – – 8
ADA Citrate
3.71 3.32
191 mM 471 mM
1.24 1.03
32 1.3
5,50 -Dinitro-BAPTA
2.15
7 mMb
–
–
References Martell and Smith (1974, 1977), Bers and MacLeod (1988) Martell and Smith (1974, 1977), Bers and MacLeod (1988) Tsien (1980) Tsien (1980) Grynkiewicz et al. (1985) Tsien (1980) Pethig et al. (1989) Tsien and Zucker (1986) Pethig et al. (1989) Pethig et al. (1989) Martell and Smith (1974, 1977), Bers and MacLeod (1988) Nakon (1979) Martell and Smith (1974, 1977), Bers and MacLeod (1988) Pethig et al. (1989)
a Abbreviations: CDTA, cyclohexilinedinitrilo-N-N-N0 -N0 -tetraacetic acid; EGTA, Ethylene glycol bis (b-aminoethylester) N-N-N0 -N0 -tetraacetic acid; BAPTA, 1,2-bis(o-aminophenoxy)ethane-N-N-N0 -N0 -tetraacetic acid; NTA, nitriloacetic acid; ADA, acetamidominodiacetic acid. b Measured at pH 7 and 0.1 M ionic strength.
Donald M. Bers et al.
4
these fluorescent indicators here, but they can be substituted for the buVers described (especially when the fundamental binding properties have been measured). Ethylene glycol bis(b-aminoethylether)-N,N,N0 ,N0 -tetraacetic acid (EGTA) is one of the best-known Ca2þ buVers, and it can be a reliable buVer in the range of 10 nM–1 mM [Ca2þ] at the typical intracellular pH of 7.2. However, if your goal is to make buVers in the 1–10 mM range, BAPTA (1,2-bis(o-aminophenoxy)ethane-N, N,N0 ,N0 -tetraacetic acid) or dibromo-BAPTA (Br2-BAPTA) would be better choices.
B. EGTA: The Workhorse of Biological Ca2þ Chelators By far the most popular Ca2þ buVer has been EGTA. This molecule has been used extensively because its apparent dissociation constant (Kd) at pH 7 (0.4 mM) is close to intracellular Ca2þ levels and it has a much higher aYnity for Ca2þ than for Mg2þ ( 100,000 times higher around neutral pH). However, the preparation of Ca2þ buVers using EGTA is complicated by the strong pH dependence of its Ca2þ aYnity (see Fig. 1 and Table I). Thus, while the free [Ca2þ] would be about 400 nM when EGTA is half saturated with Ca2þ at pH 7, the free [Ca2þ] in this same solution would decrease by nearly 10-fold to 60 nM by simply raising the pH to 7.4! Therefore, the pH of Ca2þ buVers made with EGTA must be very carefully controlled, and the calculation of the appropriate amounts of EGTA and Ca2þ to use must be made at the desired pH. The purity of the EGTA is also a variable that can cause substantial errors, as large as 0.2 pCa units in the free [Ca2þ] (Bers, 1982; Miller and Smith, 1984).
Log K Ca ⬘ (apparent Ca2+ affinity)
0.001
0.01
8 BAPTA
7
Br2-BAPTA
6
0.1
1
Free [Ca2+] (mM) for 1 Ca : 2 ligand ratio
EGTA
9
10
5
4 6
7
8 pH
Fig. 1 The pH dependence of apparent aYnities (K0 Ca) for EGTA, BAPTA, and Br2-BAPTA at 20 C and 150 mM ionic strength.
1. A Practical Guide to the Preparation of Ca2þ BuVers
5
There are many papers explaining how to calculate the proper amounts of EGTA and Ca2þ that must be combined to obtain a given free [Ca2þ] (some are listed below). Due to the steep pH dependence and slight Mg2þ sensitivity, both pH and Mg2þ must be considered in the calculation and it is best accomplished by computer. We provide a program for such calculations and describe it below. Systematic errors in EGTA purity and pH can be a real practical problem (Bers, 1982), even with the best calculations for solution preparation. Thus, we also recommend measuring the free [Ca2þ] whenever possible (see below and Chapter 3).
C. BAPTA Family of Ca2þ BuVers Roger Tsien developed an analogue of EGTA in which the methylene links between oxygen and nitrogen atoms were replaced with benzene rings to yield a compound called BAPTA (Tsien, 1980; Fig. 2). This compound exhibits a much lower pH sensitivity and much higher rates of calcium association and dissociation. These characteristics are mainly due to the fact that BAPTA is almost completely deprotonated at neutral pH. Moreover, modifications of BAPTA have been made to provide Ca2þ buVers with a range of Kd values covering the biologically significant range of 0.1 mM–10 mM (see Table I; Pethig et al., 1989). However, one disadvantage compared with EGTA is that the BAPTA family of buVers exhibits a greater ionic strength dependence (see Figs. 3–5). In particular, increasing ionic strength from 100 to 300 mM decreases the apparent aYnity constant, K0 Ca for BAPTA or Br2-BAPTA by almost threefold, whereas the COO–
–
COO–
OOC
–
OOC
N
N O
O EGTA
COO–
–
COO–
–
OOC
OOC
N
N O
X
O
X = H; BAPTA
X
X = Br; Br2-BAPTA
Fig. 2 Structural formulas for the Ca2þ chelators EGTA (top) and BAPTA and Br2-BAPTA (bottom).
Donald M. Bers et al.
6 A
K Ca ⬘ (in 106 M−1)
3
2.5
Prediction
2 Data 1.5
1 1 B
8
15
22
29
36
Temperature (⬚C) 1.5
K ⬘Ca (in 106 M−1)
1.4 1.3 Prediction
1.2 1.1 1 0.9
Data
0.8 0.7 0
0.05
0.1 0.15 0.2 Ionic strength (M)
0.25
0.3
Fig. 3 EGTA apparent Ca2þ aYnity (K0 Ca) is influenced by temperature (A) and ionic strength (B). The experimental data in A is from Harrison and Bers (1987) at pH 7.00 and 0.19 M ionic strength and in B from Harafuji and Ogawa (1980) at pH 6.8 and 22 C. Predicted values are based on the temperature and ionic strength corrections described in the text.
EGTA aYnity is only reduced by about 30%. In contrast, raising temperature from 1 to 36 C approximately doubles the apparent aYnity of all three of the Ca2þ buVers shown in Figs. 3–5 (i.e., EGTA, BAPTA, and Br2-BAPTA).
III. Methods A. Basic Mathematical Relationships From the forgoing and the data shown in Figs. 1 and 3–5, it is clear that one needs to know quantitatively how the buVers being used are altered by the typical range of experimental conditions (e.g., pH, temperature, and ionic strength). While
1. A Practical Guide to the Preparation of Ca2þ BuVers
7
A 5
K ⬘Ca (in 106 M−1)
4.5 Prediction
4 ΔH = 0
3.5
Data
3 2.5 2 1
B
8
15 22 Temperature (⬚C)
29
36
7 6 K ⬘Ca (in 106 M−1)
Prediction 5 4 3
Data
2 1 0 0.1
0.15
0.2 0.25 Ionic strength (M)
0.3
Fig. 4 BAPTA apparent Ca2þ aYnity (K0 Ca) is influenced by temperature (A) and ionic strength (B). The experimental data is from Harrison and Bers (1987) at pH 7.00 and 0.19 M ionic strength (A) and at pH 7.00 and 22 C (B). Predicted values are based on the temperature and ionic strength corrections described in the text.
we do not want to belabor the equations, it may be useful for some readers if we lay out some of the basics. If you are not interested in the equations, you can ignore this section and the next (and still use the programs as more of a black box). We hope we have accounted for things as well as possible. In the sections above, we used Kd to talk about Ca2þ aYnity. That Kd was the apparent overall dissociation constant, which we will get back to below (see Eq. (5)). It is more traditional to set out the mathematical expressions starting with the simple definition of the Ca2þ association constant KCa KCa ¼
½CaR ½Ca½R
ð1Þ
Donald M. Bers et al.
8 A 6
K Ca ⬘ (in 105 M−1)
5 Data Prediction
4
3
2 1
8
15
22
29
36
Temperature (⬚C) B
8 7
K ⬘Ca (in 105 M−1)
6 5 4
Data
3 Prediction
2 1 0 0.1
0.15
0.2 0.25 Ionic strength (M)
0.3
Fig. 5 The eVect of temperature (A) and ionic strength (B) on the apparent Ca2þ aYnity (K0 Ca) of Br2BAPTA. The experimental data is from Harrison and Bers (1987) at pH 7.00 and 0.19 M ionic strength (A) and at pH 7.00 and 22 C (B). Predicted values are based on the temperature and ionic strength corrections described in the text.
where R is the Ca2þ buVer. This expression is not too useful directly, because we do not know any of the variables on the right side. It is generally more useful to have [Ca2þ] or bound Ca2þ ([CaR]) in terms of known quantities, like total Ca2þ ([Cat]) or total chelator ([Rt]). One of the complicating factors is also that Ca2þ buVers like EGTA or BAPTA exist in multiple unbound forms in diVerent states of protonation. Then for a tetravalent Ca2þ buVer like EGTA, the total of the nonCa2þ bound forms of the buVer is ½Rt CaR ¼ ½R þ ½HR þ ½H2 R þ ½H3 R þ ½H4 R ð2Þ
1. A Practical Guide to the Preparation of Ca2þ BuVers
9 4
where we have omitted valency for simplicity. R (or R ) is the form which binds Ca2þ most avidly and it is convenient to transform Eq. (1) to one with an apparent aYnity constant for Ca2þ (K0 Ca) for a given pH 0
KCa ¼
½CaR ½R ½Ca½R ½Rt ½CaR
ð3Þ
or using Eqs. (1) and (2) 0
KCa ¼ KCa
½R ½R þ ½HR þ ½H2 R þ ½H3 R þ ½H4 R
ð4Þ
Then it is a simple matter to show that KCa þ 3
0
KCa ¼
þ
þ 2
1 þ ½H ½KH1 þ ½H KH1 KH2 þ ½H KH1 KH2 KH3 þ ½Hþ KH1 KH2 KH3 KH4 4
ð5Þ where KH1–KH4 are the four acid association constants for the buVer. Now if we know KCa, the pH, and KH1–KH4, we can calculate K0 Ca. This K0 Ca is thus the apparent aYnity for a given [H] where pH ¼ log10 ([H]/gH), and gH is the activity coeYcient for protons under the experimental conditions (see below). This K0 Ca is the reciprocal of the dissociation constant, Kd discussed in the previous section. Eq. (3) can also be manipulated to yield 0
0
½CaR=½Ca ¼ KCa ½Rt KCa ½CaR
ð6Þ
which is the linearization for Scatchard plots of Bound/Free ([CaR]/[Ca]) versus Bound ([CaR], where slope ¼ –K0 Ca and x-intercept ¼ [Rt]). One can also solve for [CaR] obtaining the familiar Michaelis–Menten form. ½CaR ¼
½Rt 0 1 þ 1= KCa ½CaÞ
ð7Þ
Solving for free [Ca] is more complicated because we do not know [CaR] a priori, but substituting [CaR] ¼ [Cat] [Ca] we can get a quadratic solution 0
0
½Ca2 þ ð½Rt ½Cat þ 1=KCa Þ½Ca ½Cat =KCa ¼ 0
ð8Þ
Similar equations can be developed for Ca2þ binding to the protonated form (e.g., H-EGTA) which also binds Ca2þ with a lower aYnity (e.g., see Harrison and Bers, 1987). For example, when we include Ca2þ binding to the singly protonated form of EGTA (or HR3) the following term must be added to the apparent aYnity expression on the right-hand side of Eq. (5) KCa2 1=ð½H KH1 Þ þ 1 þ ½H KH2 þ ½Hþ KH2 KH3 þ ½Hþ 3 KH2 KH3 KH4 þ
þ
2
ð9Þ
Donald M. Bers et al.
10
where KCa2 is the Ca association constant for the chelator in the singly protonated form, HR. This provides some basics of the relationships for a single chelator. However, more complicated solutions have multiple equilibria (e.g., other cations that bind EGTA and other Ca binding moieties) which cannot be readily solved simultaneously in an analytical manner. It should, however, be noted that it is simpler to go from free [Ca2þ] to [Cat], especially with no Ca2þ competitors. This is because all of the chelators which might bind Ca2þ will be in equilibrium with the same free [Ca2þ]. Thus, one could simply use a series of equations like Eq. (7) for diVerent chelators if you know the values on the right-hand side. Then you can simply add up free [Ca2þ] plus the [CaR] values from the chelators to obtain the [Cat]. If free [Ca2þ] is not known (or chosen) it requires multiple versions of equations like Eq. (8) to be solved simultaneously. Thus, iterative computer programs are useful (see below). 2þ
B. Temperature, Ionic Strength, and pH Corrections While the above explains the theoretical basis for calculating the pH eVect on K0 Ca, we should clarify how we normally correct for temperature, ionic strength, and pH for the experimental conditions used. Again, those not interested in the details can skip this section. Thus, the final apparent aYnity (or K0 Ca) should include correction for temperature and ionic strength as well as pH. Indeed, both proton aYnity (KH1–KH4) and metal aYnity constants (e.g., KCa) should be adjusted for the experimental temperature and ionic strength before adjusting for pH as above.
1. Temperature Corrections The standard way to correct equilibrium constants for changes in temperature depends on knowledge of the enthalpy (DH) of the reaction. 0 0 ð10Þ log10 K ¼ log10 K þ DH 1=T 1=T =ð2:303 RÞ where temperature, T is in K, DH is in kcal/mol and R is 1.9872 10 3 kcal/ (mol K). Unfortunately, the DH values are not known for all the constants we might like. For example, for EGTA they are known for the first two acid association constants (KH1 and KH2) and the higher aYnity Ca2þ constant (KCa1). This is generally suYcient for calculations with EGTA (see Fig. 3A). However, no DH values have been reported for individual constants for BAPTA and Br2-BAPTA. Harrison and Bers (1987) measured the temperature dependence of the apparent K0 Ca for BAPTA and Br2-BAPTA. We have fit that data, varying the value of the DH for KCa. This is somewhat empirical because there is likely to also be temperature dependence of KH1–KH4. However, the data was well described using DH values (for KCa) of 4.7 and 5.53 kcal/mol for BAPTA and Br2-BAPTA, respectively (see Figs. 4 and 5). Also, since BAPTA and Br2-BAPTA are almost completely
1. A Practical Guide to the Preparation of Ca2þ BuVers
11
unprotonated already at neutral pH (see Fig. 1), the adjustments to KH1 and KH2 are less important than with EGTA. However, it should be noted that one cannot simply use the DH values reported by Harrison and Bers (1987) for the overall K0 Ca of BAPTA and Br2-BAPTA (3.32 and 4.04 kcal/mol) as suggested by Marks and Maxfield (1991). That is because the intrinsic eVect of increasing temperature on the K0 Ca (with DH ¼ 0) is to reduce the K0 Ca (due to the intrinsic temperature dependence of the ionic strength adjustment, see Fig. 4A and below). Consequently, the apparent overall DH for K0 Ca (3.32 for BAPTA) is smaller than the actual DH for KCa required (our estimate is 4.7 kcal/ mol). Additionally, Harrison and Bers (1987) found the K0 Ca for Br2-BAPTA to be somewhat higher than the value predicted by the initial values reported by Tsien (1980). We find that using a slightly higher KCa (log KCa ¼ 6.96 rather than 6.8) allowed a considerably better fit to the array of experimental data shown in Fig. 5.
2. Ionic Strength Corrections Ionic strength can also dramatically alter the K0 Ca (see Figs. 3–5). We use the procedure described by Smith and Miller (1985) with ionic equivalents (Ie) rather than formal ionic strength (Ie ¼ 0.5SCi|zi|, where Ci and zi are the concentration and valence of the ith ion). We will use the terms equivalently here. Then the expression used to adjust for ionic strength is 0
0
log10 K ¼ log10 K þ 2xyðlog10 fj log10 fj Þ
ð11Þ
where K0 is the constant after conversion, K is the constant before, x and y are the valences of cation and anion involved in the reaction. The terms log10fj and log10fj0 are adjustment terms related to the activity coeYcients for zero ionic strength and desired ionic strength, respectively. To adjust for ionic strength: log10 fi ¼
AIe 1=2 1 þ Ie 1=2
bIe
ð12Þ
where b is a constant (0.25). A is a constant which depends on temperature and the dielectric constant of the medium (e) A¼
1:8246 106 ðeT Þ3=2
ð13Þ
where T is the absolute temperature ( K) and e is the dielectric constant for water. The dielectric constant is temperature dependent and can be found from tables, but the following equation provides an excellent empirical description over the range 0–50 C. e ¼ 87:7251 þ 0:3974762T þ 0:0008253T 2
ð14Þ
Donald M. Bers et al.
12
where T is in C. Thus, there is some intrinsic temperature dependence in the ionic strength adjustment itself (see Fig. 4A, broken line). These corrections provide a reasonably good description of the influence of ionic strength on the K0 Ca in Figs. 3–5. a. Activity CoeYcient for Protons The association constants as usually reported (e.g., in Martell and Smith, 1974, 1977) are often called stoichiometric (or concentration) constants. These terms are sensible because they imply (correctly) that they are to be used with concentrations or stoichiometric amounts in chemical equilibria (e.g., as in Eq. (1)). While we routinely talk about ion concentrations in ‘‘concentration’’ or ‘‘stoichiometric’’ terms, the usual exception is pH (where pH ¼ log Hydrogen ion activity or 10 pH ¼ aH ¼ gH[Hþ]). Thus, one can simply convert pH to [Hþ] and go ahead using the ‘‘stoichiometric’’ constants at face value. That is, then everything is in concentration terms and not activity. This is the way we have done it in our programs. The alternative is to change the stoichiometric constants to ‘‘mixed’’ constants (for proton interactions, or KH1–KH4 only). Then you can still use pH (or 10 pH rather than 10 pH/gH) in your calculations. Thus, acid association constants (KH1– KH4) should be divided by the value of gH. Then you can multiply the constant by the proton activity (since they are always of the same order in the equations (see Eq. (5))). That is to say that [Hþ]KH1 ¼ ([Hþ]gH)(KH1/gH), where [Hþ]gH ¼ 10 pH. This method seems a bit more awkward, but the result is the same. The proton activity coeYcient, gH varies with both temperature and ionic strength. The empirical relationship we devised to describe this relationship is the following gH ¼ 0:145045 expðB Ie Þ þ 0:063546 expð43:97704 Ie Þ þ 0:695634 ð15Þ where B ¼ 0.522932 exp(0.0327016 T) þ 4.015942 and Ie is ionic strength and T is temperature (in C). This gives very good estimates of gH from 0 to 40 C and from 0 to 0.5 M ionic strength. This expression was sent to Alex Fabiato for use in his computer program (Fabiato, 1991). While there is a typographical error in text (the first coeYcient was erroneously 1.45045), the correct expression is in the program as it was distributed.
IV. Materials A. [Ca2þ] Measurement and Calibration Solutions
1. Measuring [Ca2þ] While we can calculate the free [Ca2þ] or [Cat] for our solutions with the computer programs to be described below, there are still many potential sources of error (e.g., contaminant Ca2þ, systematic errors in pH, impurities in
1. A Practical Guide to the Preparation of Ca2þ BuVers
13
chemicals, etc.). Thus, it is valuable to measure the free [Ca2þ] to check that the solutions are as you expected (especially for complex solutions). Ca2þ sensitive electrodes are a convenient way to do this (see Chapter 3). We normally use Ca2þ minielectrodes (as described in Chapter 3) or commercial macroelectrodes. Both can be connected to a standard pH meter, but it is best to have a meter which can read in increments of 0.1 mV. We have had good luck with Orion brand Ca2þ-electrodes and they can be stable for 6 months or so. However, they are rarely as good as the home-made minielectrodes. These minielectrodes are very easy to make and are sensitive to changes in free [Ca2þ] down to 1 nM or beyond. They do not last as long as commercial macroelectrodes, but they are extremely cheap to make (per electrode) and can be discarded if they get contaminated with protein or are exposed to radioactive molecules. One can also use fluorescent indicators, once suitably calibrated, in an analogous way. The only disadvantage there is the more limited dynamic range of these Ca2þ indicators (10-fold above and below the Kd) versus electrodes which can give linear responses over the 10 nM–1 M range.
2. Spreadsheet for Calibration Calculations Making up calibration solutions for Ca2þ-electrodes (or fluorescent indicators) is really a simpler version of the multiple equilibria problem which will be discussed below (with respect to MaxChelator), because we really only need to consider the Ca2þ-EGTA buVer system. This approach is based on the paper by Bers (1982). This method has the following general steps: 1. Calculate how much total Ca2þ (or free [Ca2þ]) is required for the desired solutions (using known constants, corrected as above). All solutions should have the same dominant ionic constituents as the solutions to be measured (e.g., 140 mM KCl, 10 mM HEPES). 2. Measure the free [Ca2þ] with a good quality Ca2þ electrode compared to free [Ca2þ] standards without EGTA (at higher [Ca2þ] where [Ca2þ] is more easily controlled). 3. Accepting (for the moment) that the values from the electrode are all correct, allows the calculation of bound Ca2þ ([CaR]) from free [Ca2þ] and total [Ca2þ]. 4. Scatchard plot analysis allows the independent measurement of the apparent K0 Ca and total [EGTA] in your solutions and experimental conditions (even with systematic errors). Note that the Scatchard plot is very sensitive and deviates from linearity at very low [Ca2þ] where Ca2þ-electrodes can become sub-Nernstian in response (see Figs. 6 and 7). 5. Using these ‘‘updated’’ values of total [EGTA] and K0 Ca you can recalculate the free [Ca2þ] in the solutions. Then you can either use the free [Ca2þ] predicted from the electrode directly or you can recalculate from the total [Ca2þ] and
For entry of pCa
K ⬘Ca calculation (see A32..G47)
Solution conditions
Initial pCa 8.5000 8.0000 7.5000 7.0000 6.5000 6.0000 5.5000 5.0000 4.5000 3.0000 3.0000 3.0000
Ca-free (nM) 3.162 10 31.623 100 316.228 1000 3162.278 10000 31622.777 1,000,000 1,000,000 1,000,000
Ca-total (mM) 0.099 0.299 0.838 1.944 3.340 4.322 4.766 4.932 5.007 5.999 5.999 5.999
10 mM 1mM 100 mM
10 mM 1mM 100 mM
2.0 3.0 4.0
pH M ionic equiv (0.5*sum |zi|Ci) ⬚C mM EGTA ml bottle M or 0.1572 mM ml 100 mM CaCl2 0.493 1.496 4.188 9.722 16.701 21.609 23.831 24.662 25.034 29.995 29.995 29.995
Avg slope= Slope (mV)= mV offset at 1 mM Ca=
V-Ca (mV) −152 −142.6 −131.5 −117.1 −101.4 −85 −70.4 −51.9 −38.1 0 0 0
B= Gamma H= Log [H] = [H] (M) = K Ca ⬘ Assn= ⬘ Discn M= K Ca " (nM)= Ca-free (M) 5.38E − 09 1.14E − 08 2.74E − 08 8.60E − 08 2.99E − 07 1.10E − 06 3.51E − 06 1.52E − 05 4.56E − 05 9.38E − 04 9.38E − 04 9.38E − 04
From regression
28.2 0.8 −29.6 28.9 29 0.8
Temperature and ionic strength correction Std cond Temp 20 I-Eq 0.100 Stoich Log K const K1 9.47 K2 8.85 K3 2.66 K4 2 KCa 10.97 KCa2 5.3 Log f 0.109225 Temp 293 A 0.507424 Epsil 80.1057
Ionic str incl T eff 0.150 Log K ⬘ 9.3576 8.7657 2.6038 1.9719 10.7453 5.1315 0.12327007 296 0.51006648 79.0197311
Final Final 23⬚ C 0.150 M Log K ⬘ K ⬘ (M) 9.3138 8.7219 2.6038 1.9719 10.6840 5.1315
2.060E + 09 5.271E + 08 4.016E + 02 9.374E + 01 4.831E + 10 1.353E + 05
Regression analysis (see I29-K35)
5.12535712 Intermed 4.954 [EGTA]tot (mM) 0.76295887 H activity coefficient 0.9980 r^2 −7.0825011 Range for linear regression for scatchard should be linear electrode/scatchard slope 8.27E − 08 6.363E + 06 Log K ⬘Ca = 6.80365 6.86839 = log K ⬘Ca from scatchard 1.572E − 07 1.35E−07 = K ⬘Ca dissociation from scatchard 157.2 135.4 nM Ca-free Ca-bound B/F Regresn Inter Recalculated (nM) (mM) line B/F mediate (nM) pCa 5.383 0.099 18319.405 35857.379 −4.86E − 03 2.75 8.561 11.355 0.299 26341.654 34376.672 −4.65E − 03 8.70 8.060 27.411 0.838 30553.384 30400.143 −4.12E − 03 27.55 7.560 85.997 1.944 22608.359 22226.140 −3.01E − 03 87.48 7.058 299.131 3.340 11165.474 11917.990 −1.61E − 03 280.25 6.552 1099.966 4.321 3928.010 4674.610 −6.32E − 04 924.58 6.034 3506.127 4.763 1358.415 1409.423 −1.87E − 04 3381.53 5.471 15232.059 4.917 322.822 268.545 −2.13E − 05 17,312.44 4.762 45563.879 4.961 108.884 −55.893 5.30E − 05 63,647.15 4.196 938455.736 5.061 5.392 −790.218 1.05E − 03 1,046,090.19 2.980 938455.736 5.061 5.392 −790.218 1.05E − 03 1,046,090.19 2.980 938455.736 5.061 5.392 −790.218 1.05E − 03 1,046,090.19 2.980
Delta H kcal/mol
Valence 2*x*y
−5.8 −5.8 0 0 −8.1 0
8 6 4 2 16 12
SE of coeff R^2 F stat Reg sum Sq
[EGTA]tot K-Ca-EGTA 4.953608052 mM 7.38567143 × 10^6/M 99.07% % pure 6.868389983 = log K Regression I/J14-: 21 Regression I/J13-: 21 Slope B/F intercept Slope B/F intercept -7385.67143 36585.72 −6516.46 32752.75 135.41 548.75 435.32 1663.88 0.9980 571.40 0.9697 2324.14 2,975 6 224 7 9.71E + 08 1.96E + 06 1.21E + 09 3.78E + 07 Scatchard plot
Electrode calibration 4.E + 04
40 0 9
8
7
6
5
4
3
2 −40 −80
Initial pCa
−120
Recalculated
−160
No EGTA
−200
pCa
3.E + 04
Bound/free
1 2 3 4 5 6 7 8 9 10 11 12
Kd=
7.2 0.15 23 5 500 1.57E-07
Electrode resp (mV)
Ca calibration
Data Regression
2.E + 04 1.E + 04 0.E + 00 0.0
2.0
4.0
6.0
−1.E + 04
Bound (mM)
Fig. 6 Excel spreadsheet used to prepare Ca2þ calibration buVers using a Ca2þ electrode. This version is used when you want to start with the pCa of the calibration solutions as input and determine how much total Ca2þ is needed to achieve the desired free [Ca2þ]. It also allows updating of the apparent K0 Ca and free [Ca2þ] in the calibration solutions. This and related spreadsheets can be freely downloaded (see text for details).
1. A Practical Guide to the Preparation of Ca2þ BuVers
15 B
A 40,000
Electrode response (mV)
0
Bound/Free (mM/mM)
Regression line
30,000
20,000
10,000
Original pCa Electrode pCa Recalc. pCa
−50
−100
−150
0 0
1
2
3
4
Bound Ca2+-EGTA (mM)
5
3
4
5
6 pCa
7
8
9
Fig. 7 Scatchard plot (A) and electrode calibration curves (B) for the spreadsheet shown in Fig. 6. The Scatchard plot allows estimation of the total [EGTA] (x-intercept) and the apparent association constant, K0 Ca (-slope). The Scatchard plot is very sensitive to the detection limit of the Ca2þ electrode. The leftmost two points in A are the lowest free [Ca2þ] in the calibration curve in B (and are not included in the regression). The three calibration curves shown are for the original (or planned pCa), the pCa predicted solely by the electrode and the pCa after recalculation, using the values determined in the Scatchard plot along with the total Ca2þ added to the buVers. In this instance, there was good agreement between the three curves, but this is not always the case (see Bers, 1982).
updated constants. The latter is necessary for the lowest free [Ca2þ] where the electrode response is becoming nonlinear ( pCa 9). We use a spreadsheet (Excel) to greatly simplify all of these steps (see Fig. 6). There are three basic versions of this spreadsheet: one for starting with free [Ca2þ] as the input (DMB-CAF-2010.xls), one for pCa as input (DMB-PCA2010.xls), and one for total Ca2þ as input (DMB-CAT-2010.xls). These can be freely downloaded from the MaxChelator site as described below. We will walk you through the use of this spreadsheet in making a series of free [Ca2þ] standards here. The fields for the input of data are shaded dark gray. For the pCa version of the spreadsheet in Fig. 6 you proceed as follows (the others versions are completely analogous): 1. Enter your solution conditions (upper left, pH, ionic strength, temperature, total [EGTA], and bottle size you will use). The K0 Ca values are then automatically adjusted for the selected temperature, pH, and ionic strength (lower left box). 2. Enter the desired pCa values. The free [Ca2þ], total [Ca2þ], and ml of 100 mM Ca2þ stock are automatically calculated (next three columns) using the adjusted K0 Ca.
Donald M. Bers et al.
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3. Enter the mV readings from a Ca2þ electrode (including values for Ca2þ standards lacking EGTA at 100 mM, 1 mM, and 10 mM [Ca2þ] and the electrode reading at 1 mM free [Ca2þ] as the ‘‘oVset’’). This is the fourth column (V-Ca) and you can choose the electrode slope (rather than assume the average). The free [Ca2þ], Ca2þ-bound to EGTA, and the bound/free (B/F) are then automatically calculated (based on the electrode response and total [Ca2þ]). 4. Those calculated values (light shaded box, yellow in downloaded file) will be subject to linear regression Scatchard analysis (automatically). The Scatchard plot and Electrode calibration curves (Fig. 6, bottom) can be inspected to check linearity. If values within the regression window are not on the linear range, they can throw oV the analysis. The top and bottom two [Ca2þ] are excluded from the regression to allow calculations of [Ca2þ] for solutions outside that range. 5. Finally, the free [Ca2þ] and pCa are automatically recalculated using the measured K0 Ca and total [EGTA] (from the auto-analysis) as well as the total Ca2þ values (last two columns). The Electrode calibration curve and Scatchard plot allow you to get an overview of the results. (Fig. 6). We routinely use this for calibration solutions for both Ca2þ-electrodes and fluorescent indicators. In addition to improving the reliability of Ca2þ calibration solutions, one of the convenient aspects of this spreadsheet is that you can see all the details of what is going on. For example, you can see that the EGTA is almost completely saturated as you get up to 10 mM free [Ca2þ]. In this range we usually believe the electrode, rather than our ability to pipette within 1% of the required volume. On the other hand, as you approach the detection limit of the electrode (e.g., pCa 9), we use the recalculated pCa values. The measured versus predicted K0 Ca, EGTA purity and [Ca2þ] can also be useful in identifying potential systematic errors or changes in your procedures.
B. Preparing BuVer Solution
1. Basic Steps in Solution Preparation There are no hard and fast rules or special tricks to make these buVers, but special care in weighing and pipetting, and common sense can help avoid some potential problems. The water should be well purified to minimize contamination with Ca2þ and other metals. We usually use water that is first distilled and then run through a water purification system containing at least one ion exchange column (e.g., Nanopure, from Barnstead). This provides water with resistivity of > 15 MOhm-cm. Starting with good water like this is important for removal of other metal contaminants as well as Ca2þ. There can also be contaminating Ca2þ and metals in the salts and chemicals used to make solutions. In the end, it is typical to find 1–3 mM free [Ca2þ] in nominally Ca2þ-free solutions. This can be checked with a Ca2þ-electrode.
1. A Practical Guide to the Preparation of Ca2þ BuVers
17
Some people include 1–2 mM TPEN, a heavy metal chelator in Ca2þ-buVer solutions. This can chelate submicromolar amounts of heavy metals, which may or may not be chelated by the dominant Ca2þ buVer. This may not be important in routine applications, but may ensure that the Ca2þ-sensitive process under study will not be altered by trace amounts of other metals. All solutions should be made and stored in clean plastic ware (careful washing and extensive rinsing in deionized water is required). Glass containers should be avoided. EGTA can leach Ca2þ out of glass leading to gradual increase in free [Ca2þ] in the solutions. We have often been able to store Ca2þ calibration solutions for more than 6 months in polypropylene bottles (provided that there is no organic substrate to foster bacterial growth). An accurate [Ca2þ] standard is important for making Ca2þ buVers. It is diYcult to make accurate [Ca2þ] using CaCl2 2H2O typically used to make physiological solutions. This is because the hydration state varies making stoichiometric weighing imprecise. CaCO3 can be more accurately weighed, but has the disadvantage that you must then drive oV the CO2 with prolonged heating and HCl, unless HCO3 is desired in the solutions (which is a weak Ca2þ buVer itself). A convenient alternative is to buy a CaCl2 standard solution and we use a 100 mM CaCl2 solution from Orion (BDH also sells an excellent 1 M CaCl2 standard). To save money, one can titrate a larger volume of CaCl2 to the same free [Ca2þ] as the Orion standard using a Ca2þ-electrode. It is also important to prepare accurate stock solutions of Ca2þ chelators. EGTA from diVerent commercial sources diVer somewhat in purity (Bers, 1982; Miller and Smith, 1984), but manufacturers provide purity estimates that help (we find that purity typically ranges from 95 to 100% of the stated purity). BAPTA has also been reported to contain 20% water by weight (Harrison and Bers, 1987), but can be dried at 150 C until the weight is constant to assure removal of water. If one measures the total buVer concentration (as described in section above) this problem can be largely obviated. We typically measure the purity of each lot of EGTA or BAPTA that we use, taking this approach. Then we often keep track on the bottle itself, so that we can confirm the value upon subsequent tests with the same batch. EGTA (in the free acid form) is also not very soluble because of the acid pH. For neutral pH solutions, it is practical to dissolve EGTA with KOH in a 1:2 stoichiometry, since at neutral pH two of the four protons on EGTA are dissociated (vs. all four for BAPTA). When Ca2þ is added to EGTA solutions, 2 mol of Hþ are released for each mole of Ca2þ bound. Thus, the pH should always be adjusted as the Ca2þ is being added or afterward. The strong pH dependence of the K0 Ca of EGTA (Fig. 1) emphasizes the importance of this point. We typically measure [Ca2þ] and pH simultaneously just before the solutions are brought up to final volume (for approximate pH adjustment) and after, for final pH adjustment (as close to the third decimal place as possible) and [Ca2þ] measurement. The solutions are also checked again later to assure consistency. The rigorous attention to pH adjustment will obviously be less crucial for the BAPTA buVers.
Donald M. Bers et al.
18
It may well be asked, why not just use BAPTA rather than EGTA? The main reason is expense, BAPTA is about 30 times more expensive. The other reason is that EGTA is the ‘‘Devil we know’’ and indeed we do know much about its chemistry (e.g., metal binding constants, DH values). For applications with small volumes of solution though, it may be quite reasonable to replace EGTA with BAPTA. The ionic strength contribution of the pH buVer should also be included in the ionic strength calculation (Ie ¼ 0.5SCi|zi|). This requires calculation of the fraction of buVer in ionized form (i.e., not protonated).
2. Potential Complications Not all of the desired constants have been determined for the metals and chelators of interest. This places some limitations on how accurately one can predict the free [Ca2þ] of a given complex solution or determine how much total Ca2þ is required to achieve a desired free [Ca2þ]. The same is true for other species of interest (e.g., Mg2þ, Mg2þ-ATP). Some Ca2þ buVers also can interact with Ca2þ in multiple stoichiometries (e.g., the low aYnity Ca2þ buVer, NTA (nitrilotriacetic acid) can form Ca2þ-NTA2 complexes). There can also be systematic errors in pH measurements (Illingworth, 1981) or purity of reagents. Purity can be estimated as described above. The pH problem is actually quite common, especially with combination pH electrodes. To put it simply, the reference junction of some electrodes (particularly with ceramic junctions) can develop junction potentials which are sensitive to ionic strength. This problem can be exacerbated when the ionic strength of the experimental solutions diVers greatly from the pH standards (typically low ionic strength phosphate pH standard buVers). A systematic error in solution pH of about 0.2 pH units is not at all uncommon. As is clear from Fig. 1, this could translate into a 0.4 error in log K0 Ca and produce a two- to threefold diVerence in free [Ca2þ] even where EGTA is at its best in terms of buVer capacity. While measuring the free [Ca2þ] with an electrode can be extremely valuable, it is not foolproof either. Ca2þ electrodes are not perfectly selective for Ca2þ (see Chapter 3). For example, the selectivity of these electrodes for Ca2þ over Mg2þ is about 30,000–100,000 (Schefer et al., 1986). This roughly corresponds to the diVerence in intracellular concentrations. Thus, a 100 nM Ca2þ solution with 1 mM Mg2þ would look to the electrode like a 110–130 nM Ca2þ solution. For the Ca2þ electrodes described in Chapter 3 (using the ETH 129 chelator), the interference by Na or K is less. For 140 mM Na or K in a 100 nM Ca2þ solution, the apparent [Ca2þ] would be only about 101 nM. Some Ca2þ buVers can also interfere with Ca2þ electrodes. Citrate, DPA (dipicolinic acid), and ADA (acetamidoiminodiacetic acid), three low aYnity Ca2þ buVers were found to interfere with Ca2þ electrode measurements, while NTA did not (Bers et al., 1991). Interestingly, citrate, DPA, and ADA (which modified electrode behavior) also modified Ca2þ channel characteristics, but NTA did not.
1. A Practical Guide to the Preparation of Ca2þ BuVers
19
When Ca2þ electrodes cannot be practically used, one may still be able to use optical indicators such as the fluorescent indicators fura-2, indo-1, Fluo-4, Fluo5N for selected [Ca2þ] ranges, or the metallochromic dyes antipyralazo III, murexide, or tetramethylmurexide for higher free [Ca2þ] (Kd 200 mM, 3.6, and 2.8 mM, respectively, Ohnishi, 1978, 1979; Scarpa et al., 1978). Of course, these indicators require calibration too. A general potential complication with Ca2þ buVers is that they may alter the very processes one is interested in studying with Ca2þ buVers. For example, EGTA and other Ca2þ-chelators have been documented to increase the Ca2þ sensitivity of the plasmalemmal and SR Ca2þ-ATPase pumps and also of Naþ/Ca2þ exchange (Berman, 1982; Sarkadi et al., 1979; Schatzmann, 1973; Trosper and Philipson, 1984). For example, 48 mM EGTA decreased the apparent KCa of Naþ/Ca2þ exchange in cardiac sarcolemmal vesicles from 20 to 5 mM Ca2þ (Trosper and Philipson, 1984). These points above are not meant to discourage one from using Ca2þ buVers, but simply to point out some of the potential problems that one might encounter. Being aware of what might occur can help troubleshoot, when things do not make sense. Clearly, the use of Ca2þ buVer solutions is essential for the understanding of Ca2þ-dependent phenomena. Our aim here is to provide helpful information. C. Software Programs While the above spreadsheet is useful for very simple Ca-EGTA or Ca-BAPTA solutions used for calibrations, it is not suYcient for more complex buVers that one typically uses experimentally (which include Mg2þ in addition to Ca2þ and multiple anionic species like ATP that bind Ca2þ and Mg2þ). Several computer programs have been described (Bers et al., 1994; Brooks and Storey, 1992; Fabiato, 1988; McGuigan et al., 1991; Schoenmakers et al.., 1992; Taylor et al., 1992), but we will focus on, MaxChelator developed by one of the authors (CWP Bers et al., 1994). We have seen above that care is needed in using Ca2þ electrodes. This is equally true for any software used to determine free metal concentrations in the presence of chelators. In both cases, careful measurement of environmental conditions is needed: temperature, pH, and ionic strength, as well as attention to the quality and accuracy of measurement of all reagents. In addition, software is aVected by the choice of stability constants, quality of the code, and the particular algorithms used, and of course, the understanding of those using the software (being dependent on personal knowledge and the ease of use of the software). Fabiato and Fabiato (1979) broke ground for average users by publishing their paper on using a hand held programmable calculator to determine free [Ca2þ] or [Mg2þ] in the presence of EGTA. Before then complicated and user unfriendly software running on main frames and mini computers was all that was available. Use of Ca2þ electrodes was also just starting and not easy for most labs to implement. The Fabiato code opened this door, but was somewhat limited. Richard Steinhardt’s lab used the Fabiato paper to write a version for the Apple 2e, and one of us (CWP) further developed this to a program known as the
Donald M. Bers et al.
20
MaxChelator series of programs, first introduced in the 1994 version of this chapter. There was no internet 16 years ago when the first edition of this chapter was presented. The compilation of useful stability and thermodynamic constants (e. g., Martell and Smith, 1974, 1977) has not grown with the explosion of biological use of Ca2þ buVers and novel Ca2þ indicators (although resources are available at the National Institute of Standards and Technology (NIST) web site http:// www.nist.gov/srd/nist46.htm). For most of these new compounds, accurate stability constants have not been determined. Further, there is some disagreement over which constants and algorithms are best. However, as implied above, it is valuable to be able to calculate appropriate stoichiometric concentrations of, for example, Ca2þ, Mg2þ, EGTA, and ATP to use in your solutions to obtain the desired free [Ca2þ] and [Mg2þ] and [Mg2þ-ATP]. On the other hand, there is no substitute for actually measuring the concentration when possible to avoid imperfections in the calculations and also systematic errors (McGuigan et al., 2007). Two commonly used programs are Chelator by Theo Schoenmakers (Schoenmakers et al., 1992) and the MaxChelator series by one of the authors (CWP). Chelator is written for DOS and has not been updated since 1992 (making it less broadly useful in 2010) as fewer computers and users run DOS programs and the user interface is dated. The MaxChelator series expanded into Windows (both 16 and 32 bit), and more recently into the web via Javascript to be more OS neutral. This website has downloadable versions of the MaxChelator suite, Chelator, and several other related tools (including the Bers’ Spreadsheets as in Fig. 6): http:// maxchelator.stanford.edu/downloads.htm
1. Ideal Software Criteria 1. First and foremost is the software has to give the correct answer or at least close enough that it does not aVect the experimental conclusions (and allows measurement verification). 2. Must be easy to use. Users should not be confused as to where to enter information or what information to enter. 3. Adaptable. There should be an easy way to enter diVerent constants and possibly even allow for diVerent methods of doing some of the calculations. 4. Source code available so the knowledge is not lost with the programmer/ researchers. No current software handles all these requirements well.
1. A Practical Guide to the Preparation of Ca2þ BuVers
21
2. Accuracy We think that the constants and algorithms for the calculations in these programs are appropriate, but the ambiguities in available fundamental constants, some nuances in their application and the systematic experimental errors discussed above conspire such that solution making by recipe is imperfect. Experience and direct [Ca2þ] measurement are the best ways to limit inaccuracies in the long run. Indeed, blind acceptance of the calculations, and a presumption that there are no systematic errors (in either the solution making, pH, temperature, or in the calculations) enhances the likelihood for inaccuracies.
3. Ease of Use and Adaptability Early versions of these programs were DOS based and developed within various labs, with user interfaces not consistent with present day expectations. Patton’s MaxChelator has attempted to maintain a user friendly interface that has evolved during the past 15 years. One can input either desired free concentrations of Ca2þ, Mg2þ, Mg-ATP to obtain the total concentrations required or vice versa, and there are simple intuitive screens for these inputs. We are not aware of a commercial program that does these calculations. To ensure adaptability in this future, code should be available as open source to maximize access for future improvements (including by others). Both Chelator and MaxChelator allow for additional chelators and sets of constants to be created or changed (a useful feature), but do not allow for their inner workings or equations to be changed. If programs were open sourced then the inner algorithms could be changed to try out diVerent ideas. Software could be ‘‘tweaked’’ and refined to hopefully overcome its limitations. Another issue for the future is whether there will be suYcient interest in the continual evolution of these software suites.
4. Other Things to be Aware of When Doing This Work In line with the aforementioned concerns, Patton et al. (2004) mentioned several precautions. First, pH control is critical (within 0.01 pH unit) especially for EGTA. Moreover, when metals bind to chelators, Hþ is released aVecting pH, and that increases the importance of appropriate pH buVer choice. Second, chelators cannot reduce free metal concentration to zero. An equilibrium is set up, and [Ca2þ] and [Mg2þ] (like [Hþ]) are always finite. If proteins or other moieties in your system have higher aYnity for the metal than your chelator, it can complicate chelator eVectiveness. Contamination with Ca2þ is almost always present. Third, select the right chelator for metal concentrations of interest. Just like pH buVers which work in a range of 0.5 pH units, chelators work in a range of 0.5pKd ( 0.3–3Kd). Using too high Kd allows contaminant Ca2þ to strongly influence [Ca2þ] at the low levels, while too low Kd will result in saturation and loss of
Donald M. Bers et al.
22
buVering near the higher end. Note that the lower aYnity buVers typically have higher oV-rates and thus equilibrate faster and damp rapid [Ca2þ] spikes more eVectively.
5. Why Use Software and Where to Get MaxChelator? Despite all the caveats, using software to calculate free [Ca2þ], [Mg2þ], and [MgATP] is necessary to have a reasonable chance of getting the solutions right. And we think that MaxChelator is a useful tool in this regard. More information and downloads (free) are available at http://maxchelator.stanford.edu/. Whenever practical, it is also highly desirable to measure the [Ca2þ] using either electrodes or fluorescent Ca2þ indicators to confirm the predictions and check for reproducibility. These measurements are less practical for other metals (even Mg2þ) or anions, for which electrodes and fluorescent indicators are less available.
6. MaxChelator for Windows The earliest MaxChelator eVort was a DOS program which was then moved to Windows (Winmaxc), and the latest version is posted at the above website. The current Windows version allows visualization in two or three dimensions, some of the key factors that aVect the result. The source code is hundreds of pages long and is complied under the Delphi (Visual Pascal) environment (not posted). The files of constants are editable using a text editor and any number of files of constants can be maintained. However, the algorithms used to calculate the eVects of temperature and ionic concentration are hidden, limiting the flexibility of this version. On the other hand, it is straightforward to use and multiple metals and chelators can be easily used together (e.g., Ca2þ, Mg2þ, Ba2þ, BAPTA, Br2BAPTA, and ATP).
7. Javascript Web Versions Not everyone wants to use windows software, so the algorithms have been ported to Javascript which runs on all platforms that have a browser with Javascript enabled (with syntax similar to C programming language). One limitation is that the math libraries for interpreted Javascript are not as accurate as those for compiled programs (and rounding errors can create limitations, especially with simultaneous use of multiple metals and chelators). Some people also disable Javascript because of the fear of malware. An advantage of Javascript, besides running on most computers, is that the source code is readily accessible and can be saved, edited, and then run on any machine. If the result is an improvement, it can be shared. Another advantage is the simple user interface. Everything is in front of you all the time, and it is very easy and intuitive to change pH, temperature, ionic concentration, or metal/ chelator concentrations. Several variants are available for either online calculations or download. Some are simple binary Ca-EGTA or Mg-ATP calculators like
1. A Practical Guide to the Preparation of Ca2þ BuVers
23
the one in the screenshot below. One simply chooses the calculation type at the top, enter the temperature, pH, and ionic strength (line 2) and the two known Ca-EGTA concentrations. Not only are the traditional find free Ca2þ and find total Ca2þ calculations performed, but also the occasionally useful find total EGTA given the free and total (or free and bound) Ca2þ levels can be performed.
There are also the slightly more complex versions for Ca–Mg-ATP-EGTA equilibria. Finally, there is the more comprehensive version (Web MaxC) that allows any combination of cations (Al3þ, Ba2þ, Ca2þ, Cd2þ, Cu2þ, Fe2þ, Mg2þ, Sr2þ, Zn2þ) and 12 diVerent chelators (including EGTA, BAPTA, Br2BAPTA, EDTA, ATP, ADP, and citrate), but the simplicity and functionality are the same as the simple CaEGTA version above. There are also versions posted that use the Schoenmakers constants and conditions and other versions will be posted as they are written. These programs can thus be helpful in designing solutions with particular free ion concentrations, but should be used with understanding of the limitations.
V. Discussion and Summary It is important to be able to prepare solutions with buVered [Ca2þ], and often these solutions are complicated by multiple equilibria, and theoretical and practical limitations. Here we have discussed some of the basic principles that are involved, several key factors that complicate the process and provide some practical tools and advice to increase the probability that one can make the desired solution. However, neither the calculations nor the solution preparation nor measurement are foolproof. One must be alert to some of the potential caveats, and make independent measurements when possible. Often it is useful to make a very careful set of calibration standards at a selected ionic strength, temperature, and pH, using simpler solutions (e.g., containing simple Ca-EGTA buVers) for standardization of either a Ca2þ electrode or fluorescent Ca2þ indicator (as in Fig. 6). Note also that there are [Ca2þ] solution sets
Donald M. Bers et al.
24
sold commercially for this purpose, but they may not mimic your preferred conditions (and we have not used them). Once your electrode or fluorescent indicator is calibrated, you can use it to measure [Ca2þ] in more complex solutions, where solution predictions are less reliable. These more complex solutions could be a series of solutions of diVerent [Ca2þ] or [Mg-ATP], for example, to activate skinned muscle fiber contraction, expose to permeabilized cells, dialyze into cells via patch pipettes or use directly in biochemical assays in vitro. This is certainly a rational and practical approach. One practical caveat is that the aYnity of most fluorescent Ca2þ indicators changes (usually decreases two- to fourfold) in the cellular environment versus in protein-free solutions (Harkins et al. 1993; HoveMadsen and Bers, 1991; Konishi et al., 1988; Uto et al., 1991) and this seems to be due to the interaction of the indicators with cellular proteins (which can be mimicked in vitro). So precise control and measurement of [Ca2þ]i in cells are both very diYcult to fully achieve. On the other hand, the importance of [Ca2þ] makes it important to measure and try to control [Ca2þ] as best one can. Awareness of the limitations may seem daunting, but should not dissuade one from these valuable experiments. Even relative [Ca2þ] changes and imperfect control or measurement of [Ca2þ] are of value in understanding these processes. Acknowledgements This work was supported by a grant from the National Institutes of Health (HL30077).
References Berman, M. C. (1982). Stimulation of calcium transport of sarcoplasmic reticulum vesicles by the calcium complex of ethylene glycol bis(b-aminoethyl ether)-N,N0 ,-tetraacetic acid. J. Biol. Chem. 257, 1953–1957. Bers, D. M. (1982). A simple method for the accurate determination of free [Ca] in Ca-EGTA solutions. Am. J. Physiol. 242, C404–C408. Bers, D. M., and MacLeod, K. T. (1988). Calcium chelators and calcium ionophores. In ‘‘Handbook of Experimental Pharmacology,’’ (P. F. Baker, ed.), Vol. 83, pp. 491–507. Springer-Verlag, Berlin. Bers, D. M., Hryshko, L. V., Harrison, S. M., and Dawson, D. (1991). Citrate decreases contraction and Ca current in cardiac muscle independent of its buVering action. Am. J. Physiol. 260, C900–C909. Bers, D. M., Patton, C. W., and Nuccitelli, R. (1994). A practical guide to the preparation of Ca buVers. Methods Cell Biol. 40, 3–29. Brooks, S. P. J., and Storey, K. B. (1992). Bound and determined: A computer program for making buVers of defined ion concentrations. Anal. Biochem. 201, 119–126. Fabiato, A. (1988). Computer programs for calculating total from specified free or free from specified total ionic concentrations in aqueous solutions containing multiple metals and ligands. Methods Enzymol. 157, 378–417. Fabiato, A. (1991). Ca2þ buVering: computer programs and simulations. In ‘‘Cellular Calcium: A Practical Approach,’’ (J. G. McCormack, and P. H. Cobbold, eds.), pp. 159–176. New York, Oxford University Press. Fabiato, A., and Fabiato, F. (1979). Calculator programs for computing the composition of the solutions containing multiple metals and ligands used for experiments in skinned muscle cells. J. Physiol. Paris 75, 463–505.
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Good, N. E., Winget, G. D., Winter, W., Connolly, T. N., Izawa, S., and Singh, R. M. M. (1966). Hydrogen ion buVers for biological research. Biochemistry 5, 467–477. Grynkiewicz, G., Poenie, M., and Tsien, R. Y. (1985). A new generation of Ca indicators with greatly improved fluorescence properties. J. Biol. Chem. 260, 3440–3450. Harafuji, H., and Ogawa, Y. (1980). Re-examination of the apparent binding constant of ethlene glycol bis(b-aminoethyl ethylene)-N,N,N0 ,N0 -tetraacetic acid with calcium around neutral pH. J. Biochem. 87, 1305–1312. Harkins, A. B., Kurebayashi, N., and Baylor, S. M. (1993). Resting myoplasmic free calcium in frog skeletal muscle fibers estimated with fluo-3. Biophys. J. 65, 865–881. Harrison, S. M., and Bers, D. M. (1987). The eVect of temperature and ionic strength on the apparent Ca-aYnity of EGTA and the analogous Ca-chelators BAPTA and dibromo-BAPTA. Biochim. Biophys. Acta 925, 133–143. Hove-Madsen, L., and Bers, D. M. (1991). Indo-1 binding in permeabilized myocytes alters its spectral and Ca binding properties. Biophys. J. 63, 89–97. Illingworth, J. A. (1981). A common source of error in pH measurements. Biochem. J. 195, 259–262. Konishi, M., Olson, A., Hollingworth, S., and Baylor, S. M. (1988). Myoplasmic binding of fura2 investigated by steady-state fluorescence and absorbance measurements. Biophys. J. 54, 1089–1104. Marks, P. W., and Maxfield, F. R. (1991). Preparation of solutions with free calcium concentration in the nanomolar range using 1, 2-bis(o-aminophenoxy)ethane-N,N,N0 ,N0 -tetraacetic acid. Anal. Biochem. 193, 61–71. Martell, A. E., and Smith, R. M. (1974). Critical Stability Constants, Vol. 1. Plenum, New York. Martell, A. E., and Smith, R. M. (1977). Critical Stability Constants, Vol. 3. Plenum, New York. McGuigan, J. A. S., Lu¨thi, D., and Buri, A. (1991). Calcium buVer solutions and how to make them: A do it yourself guide. Can. J. Physiol. Pharmacol. 69, 1733–1749. McGuigan, J. A., Kay, J. W., Elder, H. Y., and Lu¨thi, D. (2007). Comparison between measured and calculated ionised concentrations in Mg2þ/ATP, Mg2þ/EDTA and Ca2þ/EGTA buVers; influence of changes in temperature, pH and pipetting errors on the ionised concentrations. Magnes. Res. 20, 72–81. Miller, D. J., and Smith, G. L. (1984). EGTA purity and the buVering of calcium ions in physiological solutions. Am. J. Physiol. 246, C160–C166. Nakon, R. (1979). Free metal ion depletion by Good’s buVers. Anal. Biochem. 95, 527–532. Ohnishi, S. T. (1978). Characterization of the murexide method: Dual wavelength spectrophotometry of cations under physiological conditions. Anal. Biochem. 85, 165–179. Ohnishi, S. T. (1979). A method of estimating the amount of calcium bound to the metallochromic indicator arsenazo III. Biochim. Biophys. Acta 586, 217–230. Patton, C., Thompson, S., and Epel, D. (2004). Some precautions in using chelators to buVer metals in biological solutions. Cell Calcium 35, 427–431. Pethig, R., Kuhn, M., Payne, R., Adler, E., Chen, T.-H., and JaVe, L. F. (1989). On the dissociation constants of BAPTA-type calcium buVers. Cell Calcium 10, 491–498. Sarkadi, B., Shubert, A., and Gardos, G. (1979). EVect of Ca-EGTA buVers on active calcium transport in inside-out red cell membrane vesicles. Experientia 35, 1045–1047. Scarpa, A., Brinley, F. J., and Dubyak, G. (1978). Antipyralazo III, a middle range Ca2þ metallochromic indicator. Biochemistry 17, 1378–1386. Schatzmann, H. J. (1973). Dependence on calcium concentrations and stoichiometry of the calcium pump in human red cells. J. Physiol. 235, 551–569. Schefer, U., Ammann, D., Pretsch, E., Oesch, U., and Simon, W. (1986). Neutral carrier based Ca2þselective electrode with detection limit in the subnanomolar range. Anal. Chem. 58, 2282–2285. Schoenmakers, T. J., Visser, G. J., Flik, G., and Theuvenet, A. P. (1992). CHELATOR: an improved method for computing metal ion concentrations in physiological solutions. Biotechniques 870–874, 876–879. Smith, G. L., and Miller, D. J. (1985). Potentiometric measurements of stoichiometric and apparent aYnity constants of EGTA for protons and divalent ions including calcium. Biochim. Biophys. Acta 839, 287–299.
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Donald M. Bers et al. Taylor, R. B., Trimble, C., Valdes, J. J., Wayner, M. J., and Chambers, J. P. (1992). Determination of free calcium. Brain Res. Bull. 29, 499–501. Trosper, T. L., and Philipson, K. D. (1984). Stimulatory eVect of calcium chelators on Naþ–Ca2þ exchange in cardiac sarcolemmal vesicles. Cell Calcium 5, 211–222. Tsien, R. Y. (1980). New calcium indicators and buVers with high selectivity against magnesium and protons: design, synthesis and properties of prototype structures. Biochemistry 19, 2396–2404. Tsien, R. Y., and Zucker, R. S. (1986). Control of cytoplasmic calcium with photolabile tetracarboxylate 2-nitrobenzhydrol chelators. Biophys. J. 50, 843–853. Uto, A., Arai, H., and Ogawa, Y. (1991). Reassessment of fura-2 and the ratio method for determination ofintracellular Ca2þ concentrations. Cell Calcium 12, 29–37.
CHAPTER 2
Photorelease Techniques for Raising or Lowering Intracellular Ca2þ Robert Zucker Molecular and Cell Biology Department University of California at Berkeley Berkeley, California, USA
Abstract I. Introduction II. Nitr Compounds A. Chemical Properties B. Calculating [Ca2þ]i Changes in Cells III. DM-Nitrophen A. Chemical Properties B. Calculating Changes in [Ca2þ]i IV. Diazo Compounds A. Chemical Properties B. Calculating EVects of Photolysis V. Introduction into Cells VI. Light Sources VII. Calibration VIII. Purity and Toxicity IX. Biological Applications A. Ion Channel Modulation B. Muscle Contraction C. Synaptic Function D. Other Applications X. Conclusions References
METHODS IN CELL BIOLOGY, VOL. 99 Copyright 2010, Elsevier Inc. All rights reserved.
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0091-679X/10 $35.00 DOI: 10.1016/S0091-679X(10)99002-X
28
Robert Zucker
Abstract The quantitative manipulation of intracellular calcium concentration ([Ca2þ]i) is a valuable instrument in the modern cell biologists’ toolbox for unraveling the many cell processes controlled by calcium. I summarize here the major classes of photosensitive calcium chelators used to elevate or reduce [Ca2þ]i, with an emphasis on their physicochemical properties and methods of calculating magnitudes and kinetics of eVects on [Ca2þ]i of flashes and steady light, in order to encourage the choice of the best substance for particular applications. The selection and calibration of appropriate light sources, and procedures for introducing the chelators into cells, spatially restricting [Ca2þ]i changes, and measuring the profiles of [Ca2þ]i changes imposed by photolysis, are also described. The final section describes a selection of biological applications.
I. Introduction Photolabile Ca2þ chelators, sometimes called caged Ca2þ chelators, are used to control [Ca2þ]i in cells rapidly and quantitatively. A beam of light is aimed at cells filled with a photosensitive substance that changes its aYnity for binding Ca2þ. Several such compounds have been invented that allow the eVective manipulation of [Ca2þ]i in cells. These compounds oVer tremendous advantages over the alternative methods of microinjecting Ca2þ salts, pharmacologically releasing Ca2þ from intracellular stores, or increasing cell membrane permeability to Ca2þ using ionophores, detergents, electroporation, fusion with micelles, or activation of voltage-dependent channels, in terms of specificity of action, repeatability and reliability of eVect, maintenance of cellular integrity, definition of spatial extent, and rapidity of eVect, all combined with the ability to maintain the [Ca2þ]i change for suYcient time to measure its biochemical or physiological consequences. Only photosensitive chelators allow the concentration of Ca2þ in the cytoplasm of intact cells to be changed rapidly by a predefined amount over a selected region or over the whole cell. Since loading can precede photolysis by a substantial amount of time, cells can recover from the adverse eVects of the loading procedure before the experiments begin. The ideal photosensitive Ca2þ chelator does not exist, but would have the following properties. 1. The compound could be introduced easily into cell, by microinjection or by loading a membrane-permeating derivative that would be altered enzymatically to an impermeant version trapped in cells. 2. The compound could be loaded with Ca2þ to such a level that the unphotolyzed form would buVer the [Ca2þ]i to near the normal resting level, so its introduction into cells would not perturb the resting Ca2þ level. Additionally, by adjusting the Ca2þ loading or selecting chelator variants, the initial resting Ca2þ level could be set to somewhat higher or lower than the normal resting concentration.
2. Photorelease Techniques for Raising or Lowering Intracellular Ca2þ
29
3. The chelator should be chemically and photolytically stable. 4. Photolysis by a bright flash of light should allow rapid changes in the free Ca2þ level; this characteristic requires rapid photochemical and subsequent dark reactions of the chelator. 5. Photolysis should be achievable with biologically appropriate wavelengths, which requires a high quantum eYciency and absorbance at wavelengths that readily penetrate cytoplasm but cause little biological damage, that is, that are not highly ionizing. For the chelator to be protected from photolysis by light needed to view the preparation would also be useful. 6. The photoproducts, or postphotolysis buVer mixture, should continue to buVer Ca2þ, and so hold it at the new level in the face of homeostatic pressure from membrane pumps and transport processes. 7. Neither the unphotolyzed chelator nor its photoproducts should be toxic, but rather should be inert with respect to all ongoing cellular molecular and physiological processes. Three classes of compounds, the nitr series, DM-nitrophen, and the diazo series, share enough of these properties to have generated intense interest and widespread popularity, and form the subjects of this review. Numerous more general reviews of photolabile or caged compounds, which contain some information on photolabile Ca2þ chelators, have appeared (Adams and Tsien, 1993; Gurney, 1993; Kao and Adams, 1993; Kaplan and Somlyo, 1989; McCray and Trentham, 1989; Ogden, 1988; Parker, 1992; Walker, 1991). Reviews focused more on photosensitive Ca2þ chelators may also be consulted (Ashley et al., 1991a; Ellis-Davies, 2003; Gurney, 1991; Kaplan, 1990).
II. Nitr Compounds A. Chemical Properties The first useful class of photosensitive Ca2þ chelators was developed by Roger Tsien. This nitr class of compounds relies on the substitution of a photosensitive nitrobenzyl group on one or both of the aromatic rings of the Ca2þ chelator 1,2-bis (o-aminophenoxy)ethane-N,N,N0 ,N0 -tetracetic acid (BAPTA) (Adams and Tsien, 1993; Adams et al., 1988; Kao and Adams, 1993; Tsien and Zucker, 1986). Light absorption results in the abstraction of the benzylic hydrogen atom by the excited nitro group and oxidation of the alcohol group to a ketone. The resulting nitrosobenzoyl group is strongly electron withdrawing, reducing the electron density around the metal-coordinating nitrogens and reducing the aYnity of the tetracarboxylate chelator for Ca2þ. In the first member of this series, nitr-2, methanol is formed as a by-product of photolysis, but in subsequent members (nitr-5, nitr-7, and nitr-8) only water is produced. Photolysis of nitr-2 is also slow (200 ms time constant). For the other nitr chelators, the dominant photolysis pathway is much faster (nitr-7, 1.8 ms; nitr-5, 0.27 ms; and nitr-8, not reported). For these reasons,
30
Robert Zucker
nitr-2 is no longer used. For the three remaining nitr compounds, photolysis is most eYcient at the absorbance maximum for the nitrobenzhydrol group, about 360 nm, although light between 330 and 380 nm is nearly as eVective. The quantum eYciency of the Ca2þ-bound form is about 1/25 (nitr-5, 0.035 and nitr-7, 0.042) and is somewhat less in the Ca2þ-free form (0.012 and 0.011). The absorbance at this wavelength is 5500 M 1 cm 1 (decadic molar extinction coeYcient) for nitr-5 and nitr-7, and 11,000 M 1 cm 1 for nitr-8. The structures of the nitr series of compounds are given in Fig. 1; and the photochemical reaction of the most popular member of this group, nitr-5, is shown in Fig. 2. The physico-chemical properties of these and other photosensitive chelators are summarized in Table I. These chelators share the advantages of the parent BAPTA chelator: high specificity for Ca2þ over Hþ and Mg2þ (Mg2þ aYnities, 5–8 mM), lack of dependence of Ca2þ aYnity on pH near pH 7, and fast buVering kinetics. One limitation is that the drop in aYnity in the nitr compounds after photolysis is relatively modest, about 40-fold for nitr-5 and nitr-7. The Ca2þ aYnity of nitr-5 drops from 0.15 to 6 mM at 120-mM ionic strength after complete photolysis. These aYnities must be reduced at higher ionic strength, roughly in proportion to the tonicity (Tsien and Zucker, 1986). By incorporating a cis-cyclopentane ring into the bridge between the chelating ether oxygens of BAPTA, nitr-7 was created
COO− COO−
COO− COO−
N
N
O
Me nitr-5
NO2 COO− COO−
O O
O
NO2
Me nitr-7
COO− COO−
N
N OMe
O
HO H
OH H NO2
O
OH H
O
COO− COO−
N O
N O
OH H
COO− COO−
N
O
O
O
COO− COO−
nitr-8
O2N
O
O
O
O
OH H NO2
nitr-9
Fig. 1 Structures of the nitr series of photolabile chelators, which release calcium on exposure to light.
2. Photorelease Techniques for Raising or Lowering Intracellular Ca2þ
CO−2
Ca − CO−2 O2C
O2C
O2C
hn
CH3
O
O
CH3 NO +
nitr-5 (high Ca2+ affinity) O
N O
NO2
O
CO−2
N
O
H
−
O2C
−
N O
Ca2+
CO2−
−
2+
N
HO
31
H2O
Photolyzed nitr-5 (low Ca2+ affinity)
O O
Fig. 2 Reaction scheme for the photorelease of nitr-5.
with significantly higher Ca2þ aYnities (54 nM, decreasing to 3 mM after photolysis at 120 mM ionic strength). To increase the change in Ca2þ-binding aYnity on photolysis, nitr-8 was created with a 2-nitrobenzyl group on each aromatic ring of BAPTA. Photolysis of each group reduces aYnity only about 40-fold, as for nitr5 and nitr-7, but photolysis of both nitrobenzyl groups reduces aYnity nearly 3000-fold, to 1.37 mM, with a quantum eYciency of 0.026. Finally, nitr-9 is a dicarboxylate 2-nitrobenzhydrol with a low Ca2þ aYnity that is unaVected by photolysis; this compound can be used to control for nonspecific eVects of the photoproducts. Initially, nitr-5 was the substance most often applied in biological experiments, largely because it was the first photolabile chelator to have most of the qualities of the ideal substance. The limited aYnity for Ca2þ of this substance in the unphotolyzed form requires that it be lightly loaded with Ca2þ when introduced into cells; otherwise, the resting [Ca2þ]i will be too high. However, the compound in a lightly loaded state contains little Ca2þ to be released on photolysis. Nitr-7 alleviates this problem with an aYnity closer to that of normal resting [Ca2þ]i, but its synthesis is more diYcult and its photochemical kinetics are significantly slower. Both compounds permit less than two orders of magnitude increase in [Ca2þ]i, generally to only the low micromolar range, and then only with very bright flashes or prolonged exposures to steady light to achieve complete photolysis. Nitr-8 permits a much larger change in [Ca2þ]i. Photolysis kinetics for this compound have not yet been reported. Neither nitr-8 nor the control compound nitr-9 is presently commercially available; nitr-5 and nitr-7 are supplied by CalBiochem (La Jolla, California).
Table I Properties of photosensitive Ca chelators Before photolysis Compound (availability)
tPhot (ms)
lmax (nm)
Q.E.Ca
Nitr-5 Nitr-7 Nitr-8 Nitr-9 Azid-1 DMnitrophen
0.27 1.8 – – <2.0 0.015;1.9
365 365 365 365 342 370
NP-EGTA DMNPE-4 NDBFEGTA Diazo-2 Diazo-4 Diazo-3
After photolysis
Q.E.free
KD-Ca (mM)
KD-Mg (mM)
KD-Ca (mM)
0.035 0.042 0.026b 0.02 1.0 0.18
0.012 0.011 – 0.02 0.9 0.18
0.145 0.054 0.5 1000 0.23 0.007c
8.5 5.4 – 10 8 0.0017c
0.002 345 – 347 0.01;0.52 330
0.23 0.09 0.7
0.23 0.09 0.7
0.08c 0.048d 0.1
9 7 15
0.134 – 0.24
0.057a 0.030a,b 0.048
0.030a 0.030a,b 0.048
2.2 5.5 89 – >1000 20
370 370 375
Before photolysis
After photolysis Ca-binding onrate
KD-Mg (mM)
e10-Ca e10-free e10-Ca e10-free (M 1 cm 1) (M 1 cm 1) (M 1 cm 1) (M 1 cm 1)
6.3 3.0 1370 1000 120 4200;89
8 5 – 10 8 2.5
5450 5780 11,000 5500 33,000 4330
5750 5540 1100 5500 27,000 4020
13,800 24,700 50,000 15,000 11,500 3150
27,300 10,000 20,000 25,000 5550 3150
0.5 0.2 – – 0.8 0.02
1000 1000 1000
9 7 15
975 5140 18,400
975 5140? 18,400?
1900 5140? 18,400?
1900 5140? 18,400?
0.017 0.01? –
0.073 0.055 >1000
3.4 2.6 20
2080 4600 2100
22,200 46,000 22,800
700 <500 700
2080 < 500 2100
0.8 0.8 0.8
tPhot, photolysis time constant; lmax, absorbance maximum of Ca-loaded compound; most eVective photolysis wavelength; Q.E.Ca,free, quantum eYciency, Ca-bound (free); KD-Ca,Mg, Ca (Mg) dissociation constant (1/aYnity) at 0.1–0.15 M ionic strength; and e10-Ca,free, decadic absorbance extinction coeYcient of Ca-bound (free) compound. a 10% of absorbed photons produce a nonphotolyzable photoproduct similar in absorbance and aYnities to unphotolyzed diazo. b For photolysis of each site. c At pH 7.2; doubles for each 0.3 pH unit reduction. d At pH 7.2; increases 2.5 for each 0.2 pH unit reduction.
2. Photorelease Techniques for Raising or Lowering Intracellular Ca2þ
33
The latest addition to the nitr-like class of compounds based on BAPTA is Azid-1 (Adams et al., 1997). This compound was derived from the high-aYnity fluorescent indicator derivative of BAPTA, fura-2, by addition of an azido substituent to fura’s benzofuran-3 position. Unlike fura-2, neither this compound nor its photoproducts are fluorescent; and unlike the other nitr compounds and the dimethoxynitrophenyl class of Ca2þ chelators (see below), it relies on the photosensitivity of an aromatic azide rather than a nitrobenzyl group. UV absorption peaking at 372 nm (342 nm for the Ca2þ-bound form) probably leads to formation of a nitrene which steals hydrogen from water to produce an amidine, which with another hydrogen converts to a nitrenium that rapidly combines with water to form an amidinium that reacts with OH to produce the final low-aYnity electronwithdrawing benzofurane-3-one photoproduct plus ammonia. Thus, photolysis absorbs one net proton and produces one molecule of ammonia for each molecule of azid-1 photolyzed, which can lead to an elevation of pHi in weakly buVered cells. This disadvantage is counterbalanced by substantial advantages. Photolysis of both Ca2þ-bound and Ca2þ-free forms of zaid-1 is phenomenally eYcient (Q.E. 1), and azid-1 is very UV-dark, absorbing at 33,000 M 1 cm 1 when Ca2þ-bound (or 27,000 M 1 cm 1 when free); these factors combine to make it 250–300 times more sensitive to light than nitr-5! Moreover, its Ca2þ-aYnity drops from 230 nM to 120 mM, on photolysis, a change that is 12 times the change in nitr5 aYnity on photolysis. Like the nitr compounds, it hardly binds Mg2þ at all (KD ¼ 8 mM), and its Ca2þ-binding ( 109 M 1 s 1) and photolysis rates (t < 2 ms) are equally rapid. In most respects, azid-1 comes closest to the ideal-caged Ca2þ compound. Unfortunately, its synthesis is quite diYcult, and it has never been commercially available; at present, apparently none exists at all. B. Calculating [Ca2þ]i Changes in Cells If nitr-5 or azid-1 is photolyzed partially by a flash of light, the reduction in Ca2þ aYnity of a portion of the chelator occurs within 0.3 ms. During this period of photolysis, low-aYnity buVer is being formed and high-aYnity buVer is vanishing while the total amount of Ca2þ remains unchanged. As the buVer concentrations change, Ca2þ ions reequilibrate among the new buVer concentrations by shifting from the newly formed low-aYnity photoproduct to the remaining unphotolyzed high-aYnity caging chelator. Since the on-rate of binding is close to the diVusion limit (as calculated from Adams et al., 1988; see also Ashley et al., 1991b), this equilibration occurs much faster than photolysis, and Ca2þ remains in quasiequilibrium throughout the photolysis period. The [Ca2þ]i in a cell rises smoothly in a step-like fashion over a period of 0.3 ms from the low level determined by the initial concentrations of total Ca2þ, and unphotolyzed chelator to a higher level determined by the final concentrations of all the chelator species after partial photolysis. At least in the case of nitr-5, [Ca2þ]i remains under the control of the low- and high-aYnity species, so the elevated Ca2þ is removed only gradually by extrusion and uptake into organelles. Thus, nitr-5 and azid-1 are well suited to
34
Robert Zucker
producing a modest but quantifiable step-like rise in response to a partially photolyzing light flash, or a gradually increasing [Ca2þ]i during exposure to steady light. Subsequent flashes cause further increments in [Ca2þ]i. These increments actually increase because, with each successive flash, the remaining unphotolyzed chelator is loaded more heavily with Ca2þ. Eventually, unphotolyzed nitr-5 or azid-1 is fully Ca2þ-bound, and subsequent flashes elevate Ca2þ by smaller increments as the amount of unphotolyzed chelator drops. If a calibrated light source is used that photolyzes a known fraction of nitr in the light path, or in cells filled with chelator and exposed either fully or partially to light, then the mixture of unphotolyzed nitr and photoproducts may be calculated with each flash (Lando` and Zucker, 1994; Lea and Ashley, 1990). The diVerent quantum eYciencies of free and Ca2þ-bound chelators must be taken into account. Simultaneous solution of the buVer equations for photolyzed and unphotolyzed chelators and native Ca2þ buVers predicts the [Ca2þ]i. For suYciently high nitr-5 concentration (above 5 mM), the native buVers have little eVect and usually may be ignored in the calculation. Further, since [Ca2þ]i depends on the proportion of chelator loaded with Ca2þ, the exact chelator concentration in the cell makes little diVerence, at least in small cells or cell processes. If the cell is large, the light intensity will drop as it passes from the front to the rear of the cell. Knowing the absorbance of cytoplasm and chelator species at 360 nm, and the chelator concentration before a flash, the light intensity and photolysis rate at any point in the cytoplasm may be calculated. A complication in this calculation is that nitr-5 photoproducts have very high absorbance (Ca2þfree photoproduct, 24,000 M 1 cm 1 and Ca2þ-bound photoproduct, 10,000 M 1 cm l) (Adams et al., 1988). As photolysis proceeds, the cell darkens and photolysis eYciency is reduced by self-screening. For azid-1 the situation is reversed: its photoproducts have much lower absorbance (11,500 and 5000 M 1 cm 1) for Ca2þ-bound and free species or 1/3 and 1/4 of the respective unphotolyzed forms. Thus light penetrates more deeply as photolysis proceeds. Regardless, with estimation of the spatial distribution of light intensity from Beer’s Law, the spatial concentrations of photolyzed and unphotolyzed chelator can be computed; from this calculation follows the distribution of the rise in [Ca2þ]i. The subsequent spatial equilibration of [Ca2þ]i can be calculated by solving diVusion equations, often in only one dimension, using the initial [Ca2þ]i and chelator distributions as the boundary conditions. EVects of endogenous buVers, uptake, and extrusion mechanisms on the rise in [Ca2þ]i can be incorporated into the calculations. Simulations of the temporal and spatial distribution of [Ca2þ]i have been devised (Zucker, 1989) and applied to experimental data on physiological eVects of [Ca2þ]i; the predicted changes in [Ca2þ]i have been confirmed with Ca2þsensitive dyes (Lando` and Zucker, 1989). Simplified and approximate models using the volume-average light intensity to calculate volume-average photolysis rate and average [Ca2þ]i changes often suYce when the spatial distribution of [Ca2þ]i is not important, for example, in small cells or processes or when estimating the change in [Ca2þ]i in a cell after diVusional equilibration has occurred.
2. Photorelease Techniques for Raising or Lowering Intracellular Ca2þ
35
III. DM-Nitrophen A. Chemical Properties Graham Ellis-Davies followed a diVerent strategy for releasing Ca2þ—by attaching a 2-nitrobenzyl group to one of the chelating amines of ethylenediaminetetraacetic acid (EDTA) to form the photosensitive chelator dimethoxynitrophenyl-EDTA or DM-nitrophen (Ellis-Davies and Kaplan, 1988; Kaplan and Ellis-Davies, 1988). Photolysis by UV light in the wavelength range 330–380 nm cleaves the DM-nitrophen with a quantum eYciency of 0.18 through multiple intermediate pathways (McCray et al., 1992) to form iminodiacetic acid and a Hþ-absorbing 2-nitrosoacetophenone derivative, with 65% of the photoproducts formed with a time constant of 15 ms and the rest with t ¼ 1.9 ms (Faas et al., 2005, 2007). A simplified reaction is shown in Fig. 3. Although DM-nitrophen binds Ca2þ with an aYnity of 7 nM at pH 7.2, Ca2þ-bound chelator forms a photoproduct-binding Ca2þ with a 4-mM aYnity, while the free form (and Mg2þ-bound forms, see below) photolyze to a 90 mM-KD photoproduct at an ionic strength of 150 mM. These values are from the most recent of the continually evolving models of DM-nitropen photolysis (Ayer and Zucker, 1999; Bollmann and Sakmann, 2005; Faas et al., 2005, 2007; Kaplan and Ellis-Davies, 1988; Neher and Zucker, 1993). Thus, complete photolysis of Ca2þ-DM-nitrophen can elevate Ca2þ over 50,000-fold, much more than photolysis of the nitr compounds or azid-1. This significant advantage is counterbalanced to some extent by the facts that the photoproducts buVer Ca2þ so weakly that the final [Ca2þ]i will be determined largely by native cytoplasmic buVers, and that the Ca2þ liberated by photolysis of DM-nitrophen will be removed more readily by extrusion and uptake pumps.
CO2−
Ca2+ −O2C −O C CO− 2 2 N N
CO2−
O
NO2
CO2− Ca2+
N
NO
CO2−
hn
CO2−
+ N H OCH3
OCH3
OCH3
OCH3
DM-nitrophen
DM-nitrophen photolysis products
(high Ca2+ affinity)
(low Ca2+ affinity)
Fig. 3 Structure of and reaction scheme for DM-nitrophen, which releases calcium on exposure to light.
36
Robert Zucker
The absorbance of Ca2þ-saturated and free DM-nitrophen is 4330 and 4020 M1 cm 1, respectively, and 0.18. A serious complication of DM-nitrophen is that it shares the cation-binding properties of its parent molecule EDTA. In particular, Hþ and Mg2þ compete for Ca2þ at the hexacoordinate-binding site. The aYnity of DM-nitrophen for Mg2þ at pH 7.2 is 1.7 mM, whereas the photoproducts bind Mg2þ with aYnities of about 2 mM. Further, both the Ca2þ- and Mg2þ-aYnities of DM-nitrophen are highly pH-dependent (Grell et al., 1989), doubling for each 0.3 units of pH increase. Thus, in the presence of typical [Mg2þ]i levels of 1–3 mM, DM-nitrophen that is not already bound to Ca2þ will be largely in the Mg2þ-bound form. Further, excess DM-nitrophen will suck Mg2þ oV ATP, which binds it substantially more weakly, compromising the ability of ATP to serve as an energy source or as a substrate for ATPases. Finally, photolysis of DM-nitrophen will lead to a jump in [Mg2þ]i as well as [Ca2þ]i, and to a rise in pH. Unless controlled by native or exogenous pH buVers, this pH change can alter the Ca2þ and Mg2þ aYnities of the remaining DM-nitrophen. In the absence of Ca2þ-loading, DM-nitrophen even may be used as a caged Mg2þ chelator (Ellis-Davies, 2006). Attributing physiological responses to a [Ca2þ]i jump, therefore, requires control experiments in which DM-nitrophen is not charged with Ca2þ. DM-nitrophen currently is sold by CalBiochem. To circumvent the problems arising from Mg2þ competing for the Ca2þ-binding site of DM-nitrophen, a second generation derivative of ethylene glycol bis(baminoethylether)-N,N,N0 ,N0 -tetraacetic acid (EGTA, which binds Mg2þ only very weakly) coupled to a light-sensitive ortho-nitrophenyl group was developed (Ellis-Davies and Kaplan, 1994). This compound, nitrophenyl-EGTA or NPEGTA, is very rapidly cleaved (t ¼ 2 ms) (Ellis-Davies, 2003) to Hþ-absorbing imidodiacetic acid photoproducts with eVective Ca2þ-KD of 1 mM, 12,500-fold higher (lower aYnity) than that of the unphotolyzed cage (80 nM) at pH 7.2, with pH-dependence similar to that of EGTA, EDTA, and DM-nitrophen. Unlike DMnitrophen, Mg2þ binding to NP-EGTA is negligible (9 mM before and after photolysis). Quantum eYciency (0.23) is similar to that of DM-nitrophen, and higher than for the nitr compounds, but less than that of azid-1. However, photolysis eYciency is seriously limited by its low absorbance (975 M 1 cm 1), only 1/6—1/4 those of the nitr compounds and DM-nitrophen, and less than 3% of azid-1’s absorbance. More recently, a dimethoxy-ortho-nitrophenyl derivative of EGTA (DMNPE-4) was introduced (Ellis-Davies and Barsotti, 2006), with somewhat higher Ca2þ aYnity (48 nM), dropping with time constants of 10 and 17 ms to 1 mM on photolysis, low Mg2þ-aYnity (7 mM), and under half the quantum eYciency (0.09) but over five times the absorbance (5140 M1cm1), thus twice the photolysis eYciency of NP-EGTA. An additional very slow phase releasing 30% of caged Ca2þ with t 667 ms was observed. Ellis-Davies’ lab has also produced a new generation of EGTA-based chelators using the novel photosensitive chromophore nitrodibenzofuran or NDBF-EGTA (Momotake et al., 2006). This compound binds Ca2þ with KD ¼ 100 nM at pH 7.2,
2. Photorelease Techniques for Raising or Lowering Intracellular Ca2þ
37
presumably with an on-rate similar to that of the other EGTA derivatives. Ca2þ aYnity drops sharply to 1 mM on photolysis with time constants of 14 and 520 ms. Quantum eYciency (0.7) and absorbance (18,400 M 1 cm 1) are extremely high, as is the change in Ca2þ aYnity (10,000-fold at pH 7.2), making this a very attractive candidate for future-caged Ca2þ research. B. Calculating Changes in [Ca2þ]i Calculating [Ca2þ]i changes on photolysis of NP-EGTA and its congeners is similar to that for the nitr compounds (if the pH dependence of binding constants is ignored), since Mg2þ binding is not an issue. Since the chelators’ Ca2þ aYnities is similar to resting cytoplasmic [Ca2þ]i levels, filling cells with a half-Ca2þ-loaded chelator will not disturb [Ca2þ]i but can release substantial amounts of Ca2þ ( 1 mM) which will be reduced about 100-fold by the cell’s endogenous buVers. However, except for NDBF-EGTA, the low absorbance usually limits flash photolysis to at most about 20%. Quantifying changes in [Ca2þ]i caused by photolysis is much more diYcult for DM-nitrophen. The initial level of [Ca2þ]i before photolysis depends upon the total concentrations of Mg2þ, Ca2þ, DM-nitrophen, ATP, and native Ca2þ buVers, because at least two buVers (DM-nitrophen and endogenous buVers) compete for Ca2þ, two buVers (ATP and DM-nitrophen) compete for Mg2þ, and, after partial photolysis, both cations also bind to the two photoproducts. Calculating equilibrium Ca2þ levels involves simultaneous solution of at least six nonlinear buVer equations (Delaney and Zucker, 1990), which is a tedious chore at best. Also, the various dissociation constants depend on ionic strength, and have been measured only at 150 mM. The high aYnity of DM-nitrophen for Ca2þ might appear to dominate the buVering of Ca2þ in cytoplasm, but this idea is misleading. A solution of DM-nitrophen that is 50% saturated with Ca2þ will hold the free [Ca2þ]i at 7 nM at pH 7.2; this action will be independent of the total DMnitrophen concentration. However, 5 mM DM-nitrophen with 2.5 mM Ca2þ and 5 mM Mg2þ will buVer free [Ca2þ]i to about 2 mM; now doubling all concentrations results in a final [Ca2þ]i of around 5 mM. Since the total [Mg2þ]i available, as free or weakly bound to ATP, is several millimolar, partially Ca2þ-loaded DMnitrophen may bring the resting Ca2þ level to a surprisingly high level. Because the solution is still buVered, this [Ca2þ] may be reduced only gradually by pumps and uptake, but eventually Ca2þ will be pumped oV the DM-nitrophen until the [Ca2þ]i is restored to its normal level. Then photolysis may lead to only tiny jumps in [Ca2þ]i. However, if a large amount of Ca2þ-loaded-DM-nitrophen is introduced into a cell relative to the total [Mg2þ]i, Ca2þ can be buVered to low levels while photolysis can release a large amount. In fact, if enough DM-nitrophen is introduced into cells with no added Ca2þ, it may gradually absorb Ca2þ from cytoplasm and intracellular stores and photolysis can produce a substantial jump in [Ca2þ]i. Therefore, both resting and the postphotolysis levels of Ca2þ may vary over very wide ranges, depending on [DM-nitrophen]i, [Mg2þ]i, and cellular [Ca2þ]i control
38
Robert Zucker
processes, all of which are diYcult to estimate or control. Thus, quantification of changes in [Ca2þ]i is not easy to achieve. The situation may be simplified by perfusing cells with Ca2þ-DM-nitrophen solutions while dialyzing out Mg2þ and mobiles endogenous buVers (Neher and Zucker, 1993; Thomas et al., 1993). Of course, this procedure will not work in studies of cell processes requiring Mg2þ-ATP or if perfusion through whole-cell patch pipettes is not possible. Another consequence of Mg2þ binding by DM-nitrophen is that cytoplasmic Mg2þ may displace Ca2þ from DM-nitrophen early in the injection or perfusion procedure, leading to a transient rise in [Ca2þ]i before suYcient DM-nitrophen is introduced into the cell (Neher and Zucker, 1993; Parsons et al., 1996; Thomas et al., 1993). Such a ‘‘loading transient’’ was accurately predicted from models of changes of the concentrations of total [Ca2þ]i, [Mg2þ]i, ATP, native buVer, and DM-nitrophen during filling from a whole-cell patch electrode (R. S. Zucker, unpublished). Since this process may have important physiological consequences, controlling it is important. The process may be eliminated largely by separating the Ca2þ-DM-nitrophen-filling solution in the pipette from the cytoplasm by an intermediate column of neutral solution [such as dilute EGTA or BAPTA] in the tip of the pipette, which allows most of the Mg2þ to escape from the cell before the DM-nitrophen begins to enter. Then most of the loading transient occurs within the tip of the pipette. One method of better controlling the change in [Ca2þ]i in DM-nitrophen experiments is to fill cells with a mixture of Ca2þ-DM-nitrophen and another weak Ca2þ buVer such as N-hydroxyethylethylenediaminetriacetic acid (HEEDTA) or l,3diaminopropan-2-ol-tetraacetic acid (DPTA) (Neher and Zucker, 1993). These tetracarboxylate Ca2þ chelators have Ca2þ aYnities in the micromolar or tens of micromolar range. If cells are filled with such a mixture without Mg2þ, the initial Ca2þ level can be set by saturating the DM-nitrophen and adding appropriate Ca2þ to the other buVer. Then photolysis of DM-nitrophen releases its Ca2þ onto the other buVer; the final Ca2þ can be calculated from the final buVer mixture in the same fashion as for the nitr compounds. Since all the constituent aYnities are highly pH dependent, a large amount of pH buVer (e.g., 100 mM) should be included in the perfusion solution, and the pH of the final solution adjusted carefully. The kinetic behavior of DM-nitrophen and the NP-EGTAs is much more complex than their equilibrium reactions. Photolysis proceeds rapidly (ts ¼ 0.2 and 2 ms for DM-nitrophen, 2 ms for NP-EGTA), but the on-rate of Ca2þ binding is much slower, about 20 mM 1 ms 1 (Ellis-Davies, 2003; Faas et al., 2005, 2007). This characteristic has particularly interesting consequences for partial photolysis of partially Ca2þ-loaded chelator. A flash of light will release some Ca2þ, which initially will be totally free. If the remaining unphotolyzed and unbound chelator concentration exceeds that of the released Ca2þ, this Ca2þ will rebind, displacing Hþ within milliseconds and producing a brief [Ca2þ]i ‘‘spike’’ (Ellis-Davies et al., 1996; Grell et al., 1989; Kaplan, 1990; McCray et al., 1992), followed by a near-step
2. Photorelease Techniques for Raising or Lowering Intracellular Ca2þ
39
fall in pH. If Mg2þ is also present, a secondary relaxation of [Ca2þ]i follows because of the slower displacement of Mg2þ from DM-nitrophen (Ayer and Zucker, 1999; Delaney and Zucker, 1990; Escobar et al., 1995, 1997). Moreover, if a steady UV source is used to photolyze DM-nitrophen, rebinding continually lags release, leading to a low (micromolar range) free [Ca2þ]i while the illumination persists. When the light is extinguished, the [Ca2þ]i drops rapidly to a low level under control of the remaining chelator (Zucker, 1993). In the case of DM-nitrophen bound to Mg2þ, achievement of equilibrium is somewhat slower (tens of milliseconds). Thus, a reversible ‘‘pulse’’ of [Ca2þ]i is generated, the amplitude of which depends on light intensity and the duration of which is controlled by the length of the illumination. This situation remains so until the remaining unphotolyzed cage becomes fully saturated with Ca2þ, whereupon [Ca2þ]i escapes from the control of the chelator, imposing a practical limit on the product of [Ca2þ]i and duration of about 0.75 mM s for DM-nitrophen. Similar kinetic considerations apply when Ca2þ is passed by photolysis from a Ca2þ cage to another buVer such as BAPTA, EGTA, HEEDTA, or DPTA. Judicious selection of buVers and buVer ratios may be used to shape this Ca2þ ‘‘spike’’ to match a hypothetical naturally occurring [Ca2þ]i(t) waveform and test its physiological consequences (Bollmann and Sakmann, 2005). If this behavior is considered undesirable, it may be avoided by using only fully Ca2þ-saturated DM-nitrophen, due to its extremely high Ca2þ-aYnity, for which rebinding to unphotolyzed chelator is impossible. Thus, the kinetic complexity of the nitrophen class of chelators can be turned to experimental advantage, greatly magnifying the flexibility of experimental [Ca2þ]i control.
IV. Diazo Compounds A. Chemical Properties In some experiments, being able to lower the [Ca2þ]i rapidly, rather than raise it, is desirable. For this purpose, caged chelators were developed. Initial attempts involved attachment of a variety of photosensitive protecting groups to mask one of the carboxyl groups of BAPTA, thus reducing its Ca2þ aYnity until restored by photolysis. Such compounds displayed low quantum eYciency (Adams et al., 1989; Ferenczi et al., 1989) and their development has not been pursued. A more successful approach (Adams et al., 1989) involved substituting one (diazo-2) or both (diazo-4) of the aromatic rings of BAPTA with an electron-withdrawing diazoketone that reduces Ca2þ aYnity, much like the photoproducts of the nitr compounds. Figure 4 shows the structures of the diazo series of chelators. Photolysis converts the substituent to an electron-donating carboxymethyl group while releasing a proton; the Ca2þ aYnity of the photoproduct is thereby increased. The reaction is illustrated in Fig. 5. Diazo-2 absorbs one photon with quantum eYciency 0.03 to increase aYnity, in 433 ms, from 2.2 mM to 73 nM at 120-mM ionic strength (or to 150 nM at 250 mM
40
Robert Zucker COO− COO−
COO− COO−
N
COO− COO−
N O
O
diazo-3
Me
N−
O
diazo-4 O
O
O N+
N
OMe
O
COO− COO−
N
N
O
diazo-2
COO− COO−
+
N
N
+
+
N−
N−
−
N N
Fig. 4 Structures of the diazo series of photolabile chelators, which take up calcium on exposure to light.
Ca2+ −
CO−2 −
CO−2
O2C
O2C
CO−2
O
O 2C
CH3 N2
HC
N2
O2C
CO−2
N
N
H2O O
O
+
−
O2C
N O
C
CH
−
N hn
O
O
CO−2
O2C
−
N
N
−
CO−2
CH3
H 2C
O
+ H+
CH3
CO−2
O Diazo-2 (low Ca2+ affinity)
Photolyzed diazo-2 (high Ca2+ affinity)
Fig. 5 Reaction scheme for the photolysis of diazo-2.
ionic strength). The absorbance maximum of the photosensitive group is 22,200 M l cm 1 at 370 nm, and drops to negligible levels at this wavelength after photolysis. A small remaining absorbance reflects formation of a side product of unenhanced aYnity and unchanged molar extinction coeYcient in 10% of the instances of eVective photon absorption. This ‘‘inactivated’’ diazo still binds Ca2þ (with some reduction in absorbance), but is incapable of further photolysis. The Ca2þ-bound form of diazo-2 has about one-tenth the absorbance of the free form, dropping to negligible levels after photolysis, with quantum eYciency of 0.057 and a time constant of 134 ms. Binding of Ca2þ to photolyzed diazo-2 is fast, with an on-rate of 8 108 M 1 s 1. Mg2þ binding is weak, dropping from 5.5 to 3.4 mM after photolysis, and pH interference is small with this class of compound. One limitation of diazo-2 is that the unphotolyzed chelator has suYcient Ca2þ aYnity that its incorporation into cytoplasm is likely to reduce resting levels to some degree, and certainly will have some eVect on [Ca2þ]i rises that occur physiologically. To obviate this problem, diazo-4 was developed with two
2. Photorelease Techniques for Raising or Lowering Intracellular Ca2þ
41
photolyzable diazoketones. Absorption of one photon increases the Ca2þ aYnity from 89 to 2.2 mM (with a 10% probability of producing a side-product with one inactivated group). Absorption of two photons (with a probability assumed to equal the square of the probability of one group absorbing one photon, and with a measured quantum eYciency of 0.015) results in further increase of the aYnity to 55 nM, a total increase of 1600-fold. This large increase in aYnity is, to some extent, oVset by the small fraction of diazo-4 that can be doubly photolyzed readily. Thus, a flash of light produces a variety of species: unphotolyzed, singly photolyzed, doubly photolyzed, singly inactivated, doubly inactivated, and singly photolyzed-singly inactivated, with a variety of transition probabilities among species (Fryer and Zucker, 1993). Unphotolyzed diazo-4 is highly absorbent (46,000 M 1 cm 1 at 371 nm for the free form; about 4600 M 1 cm 1 for the Ca2þ-bound form). The singly photolyzed species have absorbances of half these values and doubly photolyzed diazo-4 has negligible absorbance at this wavelength. Inactivation causes little change in absorbance. A third member of this series, diazo-3, has a diazoketone attached to half the cation-coordinating structure of BAPTA, and has negligible Ca2þ aYnity. On photolysis, diazo-3 produces the photochemical intermediates of diazo-2 plus a proton, and may be used to control for these eVects of photolysis of the diazo series. At one time, diazo-2 and diazo-3 (but not diazo-4) were commercially available (Molecular Probes, Eugene, and Oregon), but these stocks appear to have been exhausted. B. Calculating EVects of Photolysis As for the nitr compounds, equilibration is faster than photolysis, so a flash of light leads to a smooth step transition in the concentration of Ca2þ chelator species. If the percentage of photolysis caused by a light flash is known, the proportions of photolyzed and inactivated diazo-2, or of the six species of diazo4, can be calculated. Usually, diazo is injected without any added Ca2þ, so the eVect of photoreleased buVers is to reduce the [Ca2þ]i from its resting value. This change can be calculated only if the total Ca2þ bound to the native buVer in cytoplasm as well as the characteristics of that buVer are known. These characteristics often can be inferred from available measurements on cytoplasmic Ca2þ buVer power and the normal resting [Ca2þ]i level. The more usual application of these substances is to reduce the eVect of a physiologically imposed rise in [Ca2þ]i. In many cases, the magnitude of the source of this Ca2þ is known, as in the case of a Ca2þ influx measured as a Ca2þ current under voltage clamp or the influx through single channels estimated from single channel conductances. Also, the magnitude of the total Ca2þ increase in a response can be estimated from measured increases in [Ca2þ]; and estimates of cytoplasmic buVering. With this information, the expected eVect of newly formed diazo photoproducts on a physiological rise in [Ca2þ]i can be calculated by solving diVusion equations that are appropriate for the distribution of Ca2þ sources before and after changing the composition of the
42
Robert Zucker
mixture of buVers in the cytoplasm. Examples of such solutions of the diVusion equation exist for spherical diVusion inward from the cell surface (Nowycky and Pinter, 1993; Sala and Herna´ndez-Cruz, 1990), cylindrical diVusion inward from membranes of nerve processes (Stockbridge and Moore, 1984; Zucker and Stockbridge, 1983), diVusion from a point source (Fryer and Zucker, 1993; Stern, 1992), and diVusion from arrays of point sources (Fogelson and Zucker, 1985; Matveev et al., 2002, 2004, 2006, 2009; Pan and Zucker, 2009; Simon and Llina´s, 1985; Tang et al., 2000; Yamada and Zucker, 1992). For large cells, the spatial nonuniformity of light intensity and photolysis rate also must be considered, taking into account the absorbances of all the species of diazo and the changes in their concentration with photolysis. Like azid-1, the self-screening imposed by diazo chelators is reduced with photolysis, so successive flashes (or prolonged illumination) are progressively more eVective.
V. Introduction into Cells Photolabile chelators are introduced into cells by pressure injection from micropipettes, perfusion from whole-cell patch pipettes, or permeabilization of the cell membrane. Iontophoresis is also suitable for diazo compounds, since this procedure inserts only the Ca2þ-free form. For the caged Ca2þ substances, this method of introduction requires that the chelator load itself with Ca2þ by absorbing it from cytoplasm or intracellular stores. Filling cells from a patch pipette has the special property that, if the photolysis light is confined to the cell and excludes all but the tip of the pipette inside the cell, the pipette barrel acts as an infinite reservoir of unphotolyzed chelator. Then the initial conditions of solutions in the pipette can be restored within minutes after photolysis of the chelator in the cell. The nitr and diazo compounds are soluble at concentrations over 100 mM and DM-nitrophen is soluble at 75 mM, so levels in cytoplasm exceeding 10 mM can be achieved relatively easily, even by microinjection, making the exogenous chelator compound the dominant buVer. Nitr and diazo chelators also have been produced as membrane-permeant acetoxymethyl (AM) esters (Kao et al., 1989). Exposure of intact cells to medium containing these esters (available from CalBiochem and Molecular Probes, respectively) might result in the loading of cells with nearly millimolar concentrations, if suYcient activity of intracellular esterase is present to liberate the membraneimpermeant chelator. However, nitr-5 or nitr-7 introduced in this manner is not bound to Ca2þ, so it must sequester Ca2þ from cytoplasm, from intracellular stores, or after Ca2þ influx is enhanced, for example, by depolarizing excitable cells. The final concentration, level of Ca2þ loading, and localization of the chelator are uncertain, so this method of incorporation does not lend itself to quantification of eVects of photolysis unless cells are coloaded with a Ca2þ indicator. During loading and other preparatory procedures, the photolabile chelators may be protected from photolysis with low pass UV-blocking filters in the light path of the
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tungsten or quartz halide beams used for viewing. For more detail on these filling procedures, see Gurney (1991). Other methods of loading cells, used primarily with other sorts of caged compounds, are discussed by Adams and Tsien (1993).
VI. Light Sources Photolysis of caged Ca2þ chelators requires a bright source of near UV light. If speed is unimportant, an ordinary mercury or xenon arc lamp may be used. Mercury lamps have a convenient emission line at 366 nm. Exposure can be controlled with a shutter, using MgF-coated Teflon blades for particularly bright sources. Lamps of 100–150 W power with collimating quartz lenses provide suYcient energy to photolyze 25% of caged Ca2þ compounds in 2 s. Bulbs of larger power only generate bigger arcs, with more energy in a larger spot of similar intensity. With additional focusing, photolysis can be achieved in one-tenth the time or even less. These light sources are the appropriate choice in applications using reversible [Ca2þ]i elevation with DM-nitrophen. Fast events require the use of a laser or xenon arc flashlamp. The xenon lamps are less expensive and cumbersome; convenient commercial systems are available from Chadwick-Helmuth (El Monte, California), Rapp Optoelektronik (Hamburg, Germany), TILL Photonics (Gra¨felting, Germany), and Cairn Research (Faversham, UK). These flashlamps discharge up to 200–300 J electrical energy across the bulb to provide a pulse of 1 ms duration with up to 300 mJ energy in the 330380-nm band. The Chadwick-Helmuth unit includes only a power supply and lamp socket, so a housing with focusing optics must be constructed (see Rapp and Guth, 1988). Focusing can be accomplished with a UV-optimized elliptical reflector or with quartz refractive optics. The reflector can be designed to capture more light (i.e., have a larger eVective numerical aperture), but reflectors have greater physical distortion than well-made lenses. In practice, the reflector generates a larger spot with more total energy, but somewhat less intensity, than refractive methods. One advantage of reflectors is that they are not subject to chromatic aberration—focusing is independent of wavelength—so the UV will be focused in the same spot as visual light. This is not true of refractive lenses. To focus and aim them accurately at the sample, a UV filter must be used to block visual light and the beam must be focused on a fluorescent surface for visualization. Both types of housing are available from Rapp Optoelektronik. Using either system, photolysis rates approaching 80–90% in one flash are achievable. This rate may be reduced by imposing neutral density filters or reducing discharge energy, but the relationship between electrical and light energy is not linear and should be measured with a photometer. Flashlamps can be reactivated only after their storage capacitors have recharged, setting the minimal interval between successive maximal flashes at several seconds or more. F1ashlamps are prone to generating a number of artifacts. The discharge causes electrical artifacts that can burn out semiconductors and op amps, and reset or
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clear digital memory in other nearby equipment. Careful electrostatic shielding, wrapping inductors with paramagnetic metal, power source isolation, and using isolation circuits in trigger pulse connections to other equipment prevent most problems, which are also diminished in pulsed mercury lamps (Denk, 1997). The discharge generates a mechanical thump at the coil used to shape the current pulse through the bulb; this thump can dislodge electrodes from cells or otherwise damage the sample. Mechanical isolation of the oVending coil solves the problem. Lamp discharge also produces an air pressure pulse that can cause movement artifacts at electrodes, which can be seen to oscillate violently for a fraction of a second when videotaped during a flash. This movement can damage cells severely, especially those impaled with multiple electrodes. Small cells sealed to the end of a patch pipette often fare better against such mistreatment. To reduce this source of injury, the light can be filtered to eliminate all but the near UV. Commercial Schott filters (UG-I, UG-Il), coated to reflect infrared (IR) light, serve well for this purpose, but can cut the 330380-nm energy to 30% or less. Liquid filters to remove IR and far UV also have been described (Tsien and Zucker, 1986). Removing IR reduces temperature changes, which otherwise can exceed 1 C per flash, whereas removing far UV prevents the damaging eVects of ionizing radiation. Chlorided silver pellets and wires often used in electrophysiological recording constitute a final source of artifact. These components must be shielded from the light source or they will generate large photochemical signals. To simply aim and focus the light beam directly onto the preparation is easiest. If isolating the lamp from the preparation is necessary, the light beam may be transmitted by a fiber optic or liquid light guide, with some loss of intensity. If a microscope is being used already, the photolysis beam may be directed through the epifluorescence port of the microscope. The lamp itself, or a light guide, may be mounted onto this port. Microscope objectives having high numerical aperture and good UV transmission will focus the light quite eVectively onto a small area, which can be delimited further by a field stop aperture. With the right choice of objectives and proper optical coupling of the lamp to the light guide and the guide to the microscope port, light intensities 25 times greater than those obtained by simply aiming the focused steady lamp or flashlamp can be achieved—suYcient to half-photolyze DM-nitrophen in 25 ms of steady bright light. TILL Photonics make a xenon arc spectrophotometer (the Polychrome) with eYcient optical coupling to several commercial epifluorescence microscopes. Half reflective mirrors can be used to combine the photolysis beam with other light sources, such as those used for [Ca2þ]i measurement. However, as the optical arrangement becomes more complex, photolysis intensity inevitably decreases. The newest development in light sources is the high intensity light-emitting diode (Bernardinelli et al., 2005). This rapidly evolving and inexpensive technology can already produce 365-nm UV light at 50 mW/cm2 (with LEDs made by Prizmatix, Modi’in Ilite, Israel, e.g.), or about 20% of the intensity of a collimated xenon arc lamp. It is often important to restrict photolysis to one region of a cell (Wang and Augustine, 1995). With epi-illumination, this may be done with a field stop
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diaphragm, or by conveying the photolysis beam through a tapered quarty fiber optic filter to the cell surface (Eberius and Schild, 2001; Godwin et al., 1997). Lasers provide an alternative source of light with the advantages of a coherent collimated beam that is focused much more easily to a very small spot. Pulsed lasers such as the frequency-doubled ruby laser or the XeF excimer laser provide at least 200 mJ energy at 347 or 351 nm in 50 and 10 ns, with possible repetition rates of 1 and 80 Hz, respectively. Liquid coumarin-dye lasers, with up to 100 mJ tunable energy in the UV and pulse duration, are also available. Inexpensive nitrogen lasers providing lower pulse energies (0.25 mJ) in 5-ns pulses at 337 nm also have been developed (Engert et al., 1996) and, with appropriate focusing, might be useful. To date, lasers have found their widest application in studies of muscle contraction. More information on these laser options is contained in discussions by Goldman et al. (1984) and McCray and Trentham (1989). An adaptation of laser photolysis is the two-photon absorption technique (Denk et al., 1990). A colliding-pulse mode-locked Ti:Sapphire laser generating 100-fs pulses of 630-nm light at 80 MHz is focused through a confocal scanning microscope. Photolysis of UV-sensitive caged compounds requires simultaneous absorption of two red photons, so photolysis occurs only in the focal plane of the scanning beam. This behavior restricts photolysis to about 1 mm3 in three dimensions, but for most compounds the photolysis rate is so slow, due to their extremely limited twophoton cross sections, that several minutes of exposure are required with currently available equipment. The best results were achieved with azid-1 and NDBF-EGTA (Brown et al., 1999; DelPrincipe et al., 1999; Momotake et al., 2006), as expected from their high single photon absorbances. Azid-1 could be fully photolyzed in the two-photon focal volume with a 10-ms pulse train of 7 mW average power, with a retention time of the released Ca2þ in this volume of about 150 ms. This technique is expensive and specialized, and is still under development, but may have practical applications in revealing the precise localization within cells or subcellular organelles of fixed targets of Ca2þ action or of highly localized Ca2þ buVers. Near-UV light alone seems to have little eVect on most biological tissues, with the obvious exception of photoreceptors and the less obvious case of smooth muscle (Gurney et al., 1987). Control experiments on the eVects of light on unloaded cells, and on the normal physiological response under study, can be used to ascertain the absence of photic eVects.
VII. Calibration When designing a new optical system or trying a new caged compound, being able to estimate the rate of photolysis of the apparatus used is important. This information is necessary to adjust the light intensity or duration for the desired degree of photolysis, and to insure that photolysis is occurring at all. In principle, the fraction (F) of a substance photolyzed by a light exposure of 0 energy J can be computed from the formula e–(J–J ) ¼ (I F)/0.1, where J0 is the
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energy needed to photolyze 90% of the substance and is given by J0 ¼ hcA/Qel, where h is Planck’s constant, c is the speed of light, A is Avogadro’s number, Q is the quantum eYciency, e is the decadic molar extinction coeYcient, and l is the wavelength of the light. In practice, however, this equation is rarely useful for the following reasons. 1. Measuring the energy of the incident light on a cell accurately is diYcult, especially for light of broad bandwidth with varying intensity at diVerent wavelengths. 2. The quantum eYciency, although provided for all the photolabile chelators, is not such a well-defined quantity. The value depends critically on how it is measured, which is not always reported. In particular, the eVective quantum eYciency for a pulse of light of moderate duration (e.g., from a flashlamp) is often greater than that of either weak steady illumination or a very brief pulse (e.g., from a laser), because of the possibility of multiple photon absorptions of higher eYciency by photochemical intermediates. This phenomenon has been noted to play a particularly strong role in nitr-5 photolysis (McCray and Trentham, 1989). Thus, apparent diVerences in quantum eYciencies between diVerent classes of chelators may be mainly the results of diVerent measurement procedures. 3. Finally, the quantum eYciency is a function of wavelength, which is rarely given. A more practical and commonly adopted approach is mixing a partially Ca2þloaded photolabile chelator with a Ca2þ indicator in a solution with appropriate ionic strength and pH buVering, and measuring the [Ca2þ] change in a small volume of this solution, the net absorbance of which is suYciently small to minimize inner filtering of the photolyzing radiation. Suitable indicators include fura-2, indo-1 (Grynkiewicz et al., 1985), furaptra (Konishi et al., 1991), f1uo-3, rhod-2 (Minta and Tsien, 1989), Calcium GreenTM, OrangeTM, and CrimsonTM (Eberhard and Erne, 1991), arsenazo III (Scarpa et al., 1978), and fura-red (Kurebayashi et al., 1993). The choice depends largely on available equipment. Fura-2, indo-1, and furaptra are dual-excitation or-emission wavelength fluorescent dyes, allowing more accurate ratiometric measurement of [Ca2þ], but they require excitation at wavelengths that photolyze the photolabile Ca2þ chelators and are subject to bleaching by the photolysis light. The former problem may be minimized by using low intensity measuring light with a high sensitivity detection system. Furaptra is especially useful for DM-nitrophen, because of its lower Ca2þ aYnity. Fluo-3 and rhod-2 were designed specifically for use with photolabile chelators (Kao et al., 1989), being excited at wavelengths diVerent from those used to photolyze the chelators, but they are not ratiometric dyes and are diYcult to calibrate accurately. Calcium Green, Orange, and Crimson suVer the same limitation, but they are often used because of their fast kinetics and bright intensity, allowing the accurate tracking of fast changes in [Ca2þ]i. Arsenazo and antipyralazo are metallochromic dyes that change absorbance on binding Ca2þ,
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fortunately at wavelengths diVerent from those at which the photolabile chelators show any significant absorbance. However, these dyes are also diYcult to calibrate for absolute levels of [Ca2þ], although changes in [Ca2þ] may be determined fairly accurately. Fura-red is a ratiometric dye excited by visible light, so it might have some application in calibrating photolysis. A problem common to all the fluorescent indicators is that their fluorescent properties may be altered by the presence of photolabile chelators, which generally are used at millimolar levels whereas the indicators are present at 100 mM or less. The photolabile chelators often produce contaminating fluorescence, which also may be Ca2þ-dependent and may partially quench the fluorescence of the indicators (Hadley et al., 1993; Zucker, 1992). Thus, the indicators must be calibrated in the presence of photolabile chelator at three well-controlled [Ca2þ] levels, preferably before and after exposure to the photolysis flash, before they can be used to measure the eVects of photolysis on [Ca2þ] (Neher and Zucker, 1993). The low and high [Ca2þ] calibrating solutions may be made with excess Ca2þ or another buVer such as EGTA or BAPTA, but the intermediate [Ca2þ] solution is more diYcult to generate, since photolysis of the chelator will release some Ca2þ and change the [Ca2þ]i and pH in this solution unless it contains a very high concentration of controlling chelator and pH buVer. The calibration procedure is generally the same for any combination of chelator and indicator. A small sample of the mixture is placed in a 1-mm length of microcuvette with a 20-mm pathlength (Vitro Dynamics, Rockaway, New Jersey) under mineral oil to prevent evaporation. This cuvette is exposed repeatedly to the photolysis beam or to flashes, which should illuminate the whole cuvette uniformly, and the [Ca2þ] after each flash or exposure is measured using a microscope-based fluorescence or absorbance photometer. A small droplet of solution under mineral oil alone would work, and may be necessary if the photolysis beam is directed through the microscope and illuminates a very small area, but sometimes the fluorescent properties of the indicators are aVected by the mineral oil. This eVect would be detected in the procedure for calibrating the chelator-indicator mixture, but is best avoided using the microcuvettes, in which contact with oil is only at the edges, the fluorescence or absorbance change of which need not be measured. In some applications, such as whole-cell patch clamping of cultured cells, using the cell as a calibration chamber can be easier than any other procedure. The expected changes in [Ca2þ] depend on the chelator used. The nitr and diazo chelators should lead to a stepwise rise or fall in [Ca2þ] after each exposure; the results can be fitted to models of the chelators and their photoproducts, using their aYnities and the relative quantum eYciencies of free and bound chelators (Fryer and Zucker, 1993; Lando` and Zucker, 1989). The percentage photolysis of the chelator in response to each light exposure is the only free parameter, and is varied until the model fits the results. In the case of the high-aYnity DM-nitrophen, little rise in [Ca2þ] will occur until the total amount of remaining unphotolyzed chelator equals the total amount of Ca2þ in the solution, whereupon the [Ca2þ] will increase suddenly. Equations relating initial and final concentrations of DM-nitrophen,
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total [Ca2þ], and photolysis rate (Zucker, 1993) then may be used to calculate photolysis rate per flash or per second of steady light exposure. Most photosensitive compounds also undergo substantial absorbance changes after photolysis. These changes can be monitored during repeated exposure to the light source without a Ca2þ indicator; the number of flashes or the duration of light exposure required to reach a given percentage photolysis then can be determined. Realizing that photolysis proceeds exponentially to completion (Zucker, 1993), these data can be used to determine the photolysis rate directly. Ideally, both methods should be used to check for consistent results. A final method for determining photolysis rate is using high pressure liquid chromatography (HPLC) to separate and quantify parent chelators and photoproducts in the reaction solution after partial photolysis (Walker, 1991).
VIII. Purity and Toxicity When experiments do not work as planned, the first suspected source of error is the integrity of the photolabile chelator. DiVerent procedures have proved most useful for testing the diVerent classes of compounds. The nitr and diazo compounds undergo large absorbance changes on binding calcium and photolysis. A 100 mM solution (nominally) of the chelator is mixed with 50 mM Ca2þ in 100 mM chelexed HEPES solution (pH 7.2), and 0.3 ml is scanned in a 1-mm pathlength spectrometer. Then 1 ml 1 M K2EGTA is added to bring the [Ca2þ] to 0, and the sample is scanned again. Finally, 1 ml 5 M CaCl2 is added to provide excess Ca2þ, and a third scan is recorded. The first scan should be midway between the other two. If the first scan is closer to the excess Ca2þ scan, it is indicative of a lower than expected concentration of the chelator, probably because of an impurity. Alternatively, Ca2þ may have been present with the chelator, which may be checked by running a scan on the chelator with no added Ca2þ and comparing the result with a scan with added EGTA; they should be identical. Ca2þ free and Ca2þ-saturated chelator solutions also are scanned before and after exposure to UV light suYcient to cause complete photolysis; the spectra are compared with published figures (Adams et al., 1988, 1989; Kaplan and Ellis-Davies, 1988) to determine whether the sample was partially photolyzed at the outset. The Ca2þ aYnities of unphotolyzed and photolyzed chelators can be checked by measuring the [Ca2þ] of 50%-loaded chelators with a Ca2þ-selective electrode. The absorbance of DM-nitrophen and some related chelators is almost Ca2þ independent, so these procedures are not eVective. A solution of DM-nitrophen nominally of 2 mM concentration is titrated with concentrated CaCl2 until the [Ca2þ] measured with an ion-selective electrode suddenly increases; this change indicates the actual concentration of the chelator and gives an estimate of purity. The aYnity of the photolysis products can be measured as for the other chelators; spectra before and after photolysis indicate whether the sample was already partially photolyzed.
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Purities of 80–90% are typical for commercial samples of all the chelators, but occasional batches of 60% purity or less have been seen; these also sometimes show high degrees of toxicity. Whether such low purity is the result of poor synthesis or storage is unclear. Nitr compounds decompose detectably after only 1 day at room temperature, and exposure to ambient fluorescent lighting for 1 day causes detectable photolysis. Chelators should be shipped on dry ice and stored at 80 C in the dark; even under these conditions they do not last forever. Repeated thawing and freezing also degrades the compounds. Some of the photolabile Ca2þ chelators display a degree of biological toxicity in some preparations. Commercial samples of nitr-5 have been seen to lyse sea urchin eggs (R. S. Zucker and L. F. JaVe, unpublished results) and leech blastomeres (K. R. Delaney and B. Nelson, unpublished results) within minutes. Zucker and Haydon (1988) found that nitr-5 blocked transmitter release within 10 min of perfusion in snail neurons, whereas DM-nitrophen has no similar eVect (P. Haydon, unpublished results). These eVects are not caused by the photoproducts, since photolysis is not necessary for the problems to occur. DM-nitrophen has been observed to reduce secretion in chromaYn cells; higher chelator concentrations, photolyzed to give the same final [Ca2þ]i level, caused less secretion (C. Heinemann and E. Neher, unpublished results). The eVect was overcome partially by inclusion of glutathione in the perfusion solution, as reported for the photoproducts of other 2-nitrobenzhydrol-based caged compounds (Kaplan et al., 1978). These signs of toxicity have been observed sporadically; whether they are properties of the chelators themselves or of impurities in the samples used is unclear. The chelators have been applied successfully to a wide range of preparations without obvious deleterious results, although subtle eVects may have been missed.
IX. Biological Applications A brief synopsis of the earliest biological applications of the caged Ca2þ chelators follows along with a much more selective sampling of the more recent and extensive literature. This is included in this chapter because many of the original papers include a wealth of detail about methodology and interpretation of Ca2þ photorelease technology.
A. Ion Channel Modulation
1. Potassium and Nonspecific Cation Channels The first and still one of the major applications of photosensitive Ca2þ chelators is analysis of Ca2þ-dependent ion channels in excitable cells. In 1987, Gurney et al. first used nitr-2,-5, and-7 to activate Ca2þ-dependent Kþ current in rat sympathetic neurons. These researchers found that a single Ca2þ ion binds to the channel with rapid kinetics and 350 nM aYnity.
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The next application of the nitr chelators was in an analysis of Ca2þ-activated currents in Aplysia neurons (Lando` and Zucker, 1989). We found that Ca2þactivated Kþ and nonspecific cation currents in bursting neurons were linearly dependent on [Ca2þ]i jumps in the micromolar range, as measured by arsenazo spectrophotometry and modeling studies. Both currents relaxed at similar rates after photolysis of nitr-5 or nitr-7, reflecting diVusional equilibration of [Ca2þ]i near the front membrane surface facing the light source. Potassium current relaxed more quickly than nonspecific cation current, after activation by Ca2þ entry during a depolarizing pulse, because of the additional voltage sensitivity of the Kþ channels. This diVerence was responsible for the more rapid decay of hyperpolarizing afterpotentials than of depolarizing afterpotentials. The role of Ca2þ-activated Kþ current in shaping plateau potentials in gastric smooth muscle was explored by Carl et al. (1990). In fibers loaded with nitr-5/AM, Ca2þ photorelease accelerated repolarization during plateau potentials and delayed the time to subsequent plateau potentials, suggesting a role for changes in [Ca2þ]i and Ca2þ-activated Kþ current in slow wave generation. Another current modulated by [Ca2þ]i is the so-called M current, a muscarineblocked Kþ current in frog sympathetic neurons. Although inhibition is mediated by G-protein coupling of the receptor to phospholipase C, resting M current is enhanced by modest elevation of [Ca2þ]i (some tens of nanomolar) and reduced by greater elevation of [Ca2þ]i, which also suppresses the response to muscarine (Marrion et al., 1991). As for ventricular ICa (see below), several sites of modulation of M current by [Ca2þ]i apparently exist. In these experiments, [Ca2þ]i was elevated by photorelease from nitr-5 and simultaneously measured with fura-2. Step changes in [Ca2þ]i imposed by diazo-2 photolysis and monitored with bisfura-2 fluorescence changes have also been used to characterize the modulation of cGMP-gated ion channels by [Ca2þ]i (Rebrik et al., 2000). The after-hyperpolarization that follows spikes in rat hippocampal pyramidal neurons is caused by a class of Ca2þ-dependent Kþ channels called IAHP channels. This after-hyperpolarization and the current underlying it rise slowly to a peak 0.5 s after the end of a brief burst of spikes. Ca2þ photorelease from nitr-5 or DM-nitrophen activates this current without delay (Lancaster and Zucker, 1994), and the current may be terminated rapidly by photolysis of diazo-4 (but see conflicting results of Sah and Clements, 1999), suggesting that the delay in its activation following action potentials is caused by a diVusion delay between points of Ca2þ entry and the IAHP channels. The Ca2þ sensitivity of the mechanoelectrical transduction current in chick cochlear hair cells was studied using nitr-5 introduced by hydrolysis of the AM form (Kimitsuki and Ohmori, 1992). Elevation of [Ca2þ]i to 0.5 mM (measured with fluo-3) diminished responses to displacement of the hair bundle, and accelerated adaptation during displacement when Ca2þ entry occurred. Preventing Ca2þ influx blocked adaptation. Evidently, adaptation of this current was the result of an action of Ca2þ ions entering through the transduction channels. In guinea pig hepatocytes, noradrenaline evokes a rise in Kþ conductance after a seconds-long delay. Photorelease of Ca2þ from nitr-5 and use of caged inositol
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1,4,5-trisphosphate (caged-IP3) show that this delay arises from steps prior to or during generation of IP3 (Ogden et al., 1990), which releases Ca2þ from intracellular stores to activate Kþ current.
2. Calcium Channels The first application of DM-nitrophen was in a study of Ca2þ channels in chick dorsal root ganglion neurons (Morad et al., 1988). With divalent charge carriers, inactivation by photorelease of intracellular Ca2þ occurred within 7 ms, whereas with monovalent charge carriers a nearly instantaneous block occurred, especially when Ca2þ was released extracellularly. A similar rapid block of monovalent current through Ca2þ channels was observed in response to photorelease of extracellular Ca2þ in frog ventricular cells (Na¨bauer et al., 1989). DiVerent Ca2þbinding sites may be exposed if altered conformational states are induced in the channels by the presence of diVerent permeant ions. The regulation of Ca2þ current (ICa) in frog atrial cells by [Ca2þ]i also has been studied with nitr-5 (Charnet et al., 1991; Gurney et al., 1989). Rapid elevation of [Ca2þ]i potentiated high-voltage-activated or L-type ICa and slowed its deactivation rate when Ba2þ was the charge carrier, after a delay of several seconds. Inclusion of BAPTA in the patch pipette solution blocked the eVect of nitr-5 photolysis. The similarity of eVect of Ca2þ and cAMP and their mutual occlusion suggest a common phosphorylation mechanism. Regulation of ICa in guinea pig ventricular cells appears to be more complex (Bates and Gurney, 1993; Hadley and Lederer, 1991). A fast phase of inactivation reflects a direct action on Ca2þ channel permeation, since ICa inactivation caused by photorelease of Ca2þ from nitr-5 is independent of the phosphorylation state of the channels and does not alter gating currents. A late potentiation is also present, the magnitude of which depends on the flash intensity delivered during a depolarizing pulse, but not on the initial [Ca2þ]i level, the degree of loading of nitr-5, or the presence of BAPTA in the patch pipette. This result suggests that, during a depolarization, nitr-5 becomes locally loaded by Ca2þ entering through Ca2þ channels, and that the Ca2þ-binding site regulating potentiation is near the channel mouth. Larger [Ca2þ]i jumps elicited by photolysis of DM-nitrophen evoke greater ICa inactivation, but no potentiation, perhaps because of the more transient rise in [Ca2þ]i when DM-nitrophen is photolyzed. DM-nitrophen loaded with magnesium in the absence of Ca2þ was used to study the Mg2þ-nucleotide regulation of L-type ICa in guinea pig cardiac cells (Backx et al., 1991; O’Rourke et al., 1992). In the presence of ATP, a rise in [Mg2þ]i to 50–200 mM led to a near doubling of the magnitude of ICa. Release of caged ATP also increased ICa. Therefore, the eVect on Ca2þ channels was caused by a rise in Mg2þ-ATP. Nonhydrolyzable ATP analogs worked as well as ATP, so Mg2þ-ATP seems to modulate Ca2þ channels directly. We microinjected Aplysia neurons with nitr-5, DM-nitrophen, or diazo-4 to characterize Ca2þ-dependent inactivation of Ca2þ current (Fryer and Zucker,
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1993). Elevation of [Ca2þ]i to a few micromolar with nitr-5 caused little inactivation, but photolysis of DM-nitrophen rapidly inactivated half the ICa, presumably that in the half of the cell facing the light source. Thus, inactivation requires high [Ca2þ]i levels and occurs rapidly in all channels, even if they are closed. Experiments with diazo-4 showed that an increase in buVering power reduced the rate of inactivation of ICa modestly. DiVusion-buVer reaction simulations suggest that Ca2þ acts at a site within 25 nm of the channel mouth (see also Johnson and Byerly, 1993). B. Muscle Contraction One of the earliest applications of photolabile Ca2þ chelators was initiating muscle contraction in frog cardiac ventricular cells by photorelease of extracellular Ca2þ from DM-nitrophen (Na¨bauer et al., 1989). The strength of contraction elicited by a stepwise rise in [Ca2þ]e showed a membrane potential dependence that was indicative of entry through voltage-dependent Ca2þ channels rather than of transport by Naþ–Ca2þ exchange. Several laboratories have used caged Ca2þ chelators to study Ca2þdependent Ca2þ release from the sarcoplasmic reticulum in rat ventricular myocytes. Valdeolmillos et al. (1989) loaded cells with the AM form of nitr-5, Kentish et al. (1990) subjected saponin-skinned fibers to solutions containing Ca2þ-loaded nitr-5, and Na¨bauer and Morad (1990) perfused single myocytes with DM-nitrophen loaded with Ca2þ. Photolysis elicited a contraction blocked by ryanodine or caVeine, procedures that prevent release of Ca2þ from the sarcoplasmic reticulum, implicating Ca2þ-induced Ca2þ release, which could be confined to a portion of a fiber by localized photolysis (O’Neill et al., 1990). Gyo¨rke and Fill (1993) used Ca2þ-DM-nitrophen to show that the cardiac ryanodine receptors adapt to maintained [Ca2þ]i elevation, remaining sensitive to larger [Ca2þ]i changes and responding by releasing still more Ca2þ. In smooth muscle from guinea pig portal vein, the IP3-dependent release of Ca2þ was itself dependent upon [Ca2þ]i (Iino and Endo, 1992). Ca2þ photoreleased from DMnitrophen and measured with fluo-3 accelerated Ca2þ release from a ryanodineinsensitive, IP3-activated store. The possibility that adaptation reflected slow unbinding of Ca2þ from the channels following a flash-induced Ca2þ ‘‘spike’’ was refuted by demonstrating a rapid deactivation of channel function to a sudden drop in [Ca2þ]i imposed by diazo-2 (Ve´lez et al., 1997). Ca2þ-loaded nitr-5 was used in skinned frog and scallop muscle fibers to show that the rate-limiting step in contraction is not the time-course of the rise in [Ca2þ]i but rather the response time of the contractile machinery (Ashley et al., 1991b; Lea and Ashley, 1990). Using isolated myofibrillar bundles from barnacle muscle, Lea and Ashley (1990) showed that nitr-5 photolysis elevating [Ca2þ]i by 0.2–1.0 mM Ca2þ not only activated contraction directly and rapidly but also evoked a slower phase of contraction that was dependent on Ca2þ-induced Ca2þ release from the sarcoplasmic reticulum. Analysis of [Ca2þ]i steps imposed by DM-nitrophen or
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diazo-2 revealed kinetics of muscle contraction and relaxation steps following the binding and unbinding of Ca2þ to troponin C (Ashley et al., 1993). The first biological application of the caged chelator diazo-2 was in the study of muscle relaxation. Mulligan and Ashley (1989) showed that rapid reduction in [Ca2þ]i in skinned frog semitendinosus muscle resulted in a relaxation similar to that occurring normally in intact muscle, indicating that mechanochemical events subsequent to the fall in [Ca2þ] were rate limiting. However, Lannergren and Arner (1992) reported some speeding of isometric relaxation after photolysis of diazo-2, loaded in the AM form into frog lumbrical fibers. Lowered pH slowed relaxation to a step reduction in [Ca2þ]i (Palmer et al., 1991), perhaps accounting for a contribution of low pH to the sluggish relaxation of fatigued muscle. In contrast to frog muscle, photorelease of Ca2þ chelator caused a much faster relaxation in skinned scallop muscle than in intact fibers (Palmer et al., 1990), suggesting that, in these cells, relaxation is rate limited primarily by [Ca2þ]i homeostatic processes. C. Synaptic Function Action potentials evoke transmitter release in neurons by admitting Ca2þ through Ca2þ channels. Because of the usual coupling between depolarization and Ca2þ entry, assessing the possibility of an additional direct action of membrane potential on the secretory apparatus has been diYcult. Photolytic release of presynaptic Ca2þ by nitr-5 perfused into presynaptic snail neurons cultured in Ca2þ-free media was combined with voltage clamp of the presynaptic membrane potential to distinguish the roles of [Ca2þ]i and potential in neurosecretion (Zucker and Haydon, 1988), revealing no direct eVect of membrane potential on transmitter release. Hochner et al. (1989) injected Ca2þ-loaded nitr-5 into crayfish motor neuron preterminal axons, and used a low-[Ca2þ] medium to block normal synaptic transmission. They found that action potentials transiently accelerated transmitter release evoked by modest photolysis of nitr-5. However, Mulkey and Zucker (1991) used fura-2 to show that the extracellular solutions used by Hochner et al. (1989) failed to block Ca2þ influx through voltage-dependent Ca2þ channels. When external Ca2þ chelators or channel blockers eliminated influx completely, spikes failed to have any influence on transmitter release, even when it was activated strongly by photolysis of intracellularly injected Ca2þ-loaded DMnitrophen. Delaney and Zucker (1990) confirmed at the squid giant synapse that in a Ca2þfree medium, action potentials have no eVect on transmitter release triggered by a rise in [Ca2þ]i upon photolysis of presynaptically injected DM-nitrophen. Flash photolysis of DM-nitrophen produced a transient postsynaptic response resembling normal excitatory postsynaptic potentials. The intense phase of transmitter release was probably caused by the brief spike in [Ca2þ]i following partial photolysis of partially Ca2þ-loaded DM-nitrophen. This response began a fraction of a millisecond after the rise in [Ca2þ]i, a delay similar to the usual synaptic delay
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following Ca2þ influx during an action potential; both delays had the same temperature dependence. Thus photolysis of DM-nitrophen caused a [Ca2þ]i transient resembling that occurring normally at transmitter release sites in the vicinity of Ca2þ channels that open briefly during an action potential. After the secretory burst, a moderate phase of transmitter release persisted for 15 ms, corresponding to a relaxation in [Ca2þ]i measured with fura-2 that probably reflected slow Ca2þ displacement of Mg2þ bound to unphotolyzed DM-nitrophen. Similar responses to partial flash photolysis of lightly Ca2þ-loaded DM-nitrophen were observed at crayfish neuromuscular junctions (Lando` and Zucker, 1994). Transmitter release evoked by slow photolysis of Ca2þ-DM-nitrophen using steady illumination also has been studied at this junction (Mulkey and Zucker, 1993). The rate of quantal transmitter release, measured as the frequency of miniature excitatory junctional potentials (MEJPs), was increased 1000-fold during the illumination. Brief illumination (0.3–2 s) evoked a rise in MEJP frequency that dropped abruptly back to normal when the light was extinguished, as would be expected from the reversible rise in [Ca2þ]i that should be evoked by such illumination, which leaves most of the DM-nitrophen unphotolyzed (Zucker, 1993). Longer light exposures caused an increase in MEJP frequency that outlasted the light signal, as would be expected from the rise in resting [Ca2þ]i after photolysis of most DM-nitrophen. These experiments illustrate the utility of steady photolysis of partially Ca2þ-loaded DM-nitrophen in generating reversible changes in [Ca2þ]i in cells. At cultured snail synapses, FMRFamide inhibits asynchronous transmitter release elicited by [Ca2þ]i elevated by photolysis of presynaptic nitr-5 (Man-SonHing et al., 1989), and blocks synchronous release to partial flash photolysis of partially Ca2þ-loaded DM-nitrophen (Haydon et al., 1991). As at crayfish and squid synapses, these flash-evoked postsynaptic responses resembled the spikeevoked responses and were triggered by the spike in [Ca2þ]i that results when DMnitrophen is used in this fashion. At leech serotonergic synapses, a presynaptic Ca2þ uptake process may be activated by photolysis of presynaptic DM-nitrophen; blocking it with zimelidine or by external Naþ removal eliminated the presynaptic transport current and prolonged the postsynaptic response, uncovering a contribution of this process to the termination of transmitter release (Bruns et al., 1993). DM-nitrophen has been used extensively to probe the steps involved in exocytosis in endocrine cells. Measuring [Ca2þ]i changes with furaptra, we and others (Heinemann et al., 1994; Neher and Zucker, 1993; Thomas et al., 1993) observed— in bovine chromaYn cells and rat melanotrophs—three kinetic secretory phases in response to [Ca2þ]i steps to 100 mM, reflecting release from diVerent vesicle pools. Prior exposure to a modest [Ca2þ]i rise primed phasic responses to a subsequent step in [Ca2þ]i, indicating that [Ca2þ]i not only triggers exocytosis but also mobilizes vesicles into a docked or releasable status. After exocytosis, another [Ca2þ]i stimulus often evoked a rapid reduction in membrane capacitance signaling a [Ca2þ]i-dependent compensatory endocytosis.
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The release of synaptic transmitter by action potentials is often enhanced for about a second after short bouts of presynaptic activity (synaptic facilitation), and for much longer after sustained activity lasting minutes (posttetanic potentiation, PTP). We have used photolytic release of presynaptic Ca2þ from DM-nitrophen to induce facilitation without electrical activity (Kamiya and Zucker, 1994). Photolysis of diazo-2 or diazo-4 to terminate the [Ca2þ]i increase lingering briefly after a short spike train abolished facilitation immediately at crayfish neuromuscular junctions. PTP induced by longer stimulation, both in this preparation and in Aplysia neuronal synapses (Fischer et al., 1997), was abolished more slowly by rapid reduction of the prolonged residual [Ca2þ]i resulting from mitochondrial overload (Tang and Zucker, 1997). Thus, facilitation and PTP arise from residual [Ca2þ]i acting on distinct molecular targets diVerent from the secretory trigger, which is also activated by Ca2þ. We compared responses to [Ca2þ]i steps on flash photolyzing DMNPE-4 at weakly transmitting but strongly facilitating neuromuscular junctions to responses at strongly transmitting but depressible junctions (Millar et al., 2005), and concluded that the diVerence in response kinetics was best explained by a diVerence in the state of Ca2þ-dependent priming, such that strongly transmitting synapses were already preprimed at rest by a priming target tuned to have a higher Ca2þ-sensitivity. This led, in turn, to the development of a comprehensive model of synaptic transmission, facilitation, and depression that comprised three Ca2þ-dependent processes—vesicle mobilization to docking sites, priming of docked vesicles, and activation of membrane fusion (Pan and Zucker, 2009). We (Lando` and Zucker, 1989) and Heidelberger et al. (1994) were the first to use DM-nitrophen photolysis to characterize the Ca2þ-cooperativity of secretion at neuromuscular junctions and retinal bipolar neurons; subsequently, we (Ohnuma et al., 2001) used NP-EGTA and Kasai et al. (1999) used DM-nitrophen to show diVerences in the Ca2þ-dependence and sensitivity of peptidergic or aminergic large dense core vesicle fusion and cholinergic small clear vesicle fusion at central molluscan synapses and in PC12 cells. Hsu et al. (1996) reported that transmitter release at squid giant synapses decayed to step [Ca2þ]i increases produced by NP-EGTA photolysis; subsequent higher steps evoked more release, indicating transmitter stores had not been depleted, suggesting either an adaptation of the release process, as the authors proposed, or possibly vesicle heterogeneity in sensitivity to release, or the operation of mobilization or priming processes enabling release of previously undocked or unprimed vesicles at higher [Ca2þ]i. Caged Ca2þ photolysis has been used extensively in the last decade in many elegant experiments, especially from the laboratories of Erwin Neher and Bert Sakmann, to kinetically characterize in detail the secretory trigger for neurosecretion, primarily at the giant synapse of the calyx of Held (Bollmann and Sakmann, 2005; Bollmann et al., 2000; Felmy et al., 2003a,b; Hosoi et al., 2007; Sakaba et al., 2005; Schneggenburger and Neher, 2000; Wadel et al., 2007; Wang et al., 2004; Young and Neher, 2009). Ca2þ uncaging from DM-nitrophen has been used to probe the kinetics and cooperativity of Ca2þ binding to the secretory trigger, kinetic consequences of SNARE protein and synaptotagmin mutation, eVects of
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Ca2þ on synaptic facilitation, the dependence of release kinetics on the time-course of local [Ca2þ]i changes, heterogeneity of vesicle Ca2þ-sensitivity and release kinetics, the role of Ca2þ in mobilizing vesicles to replenished pools depleted in synaptic depression, and to compare secretion evoked by global [Ca2þ]i manipulation in uncaging to local [Ca2þ]i influx though voltage-dependent channels to address the question of distance of the secretory target from Ca2þ channels and its changes in development. Kinetic studies of the Ca2þ-dependence of secretion using DM-nitrophen have also been conduced on cochlear hair cells (Beutner et al., 2001) and photoreceptors (Duncan et al., 2010). Zoran et al. (1991) used Ca2þ photorelease to study synapse maturation. Spikeevoked transmitter release begins only several hours after cultured snail neurons contact a postsynaptic target. DM-nitrophen photolysis showed this developmental change to result from the delayed increase in Ca2þ-sensitivity of the secretory machinery. Long-term potentiation and depression (LTP and LTD) in mammalian cortical synapses are involved in cognitive processes such as memory consolidation and spatial learning. We found that a brief but strong postsynaptic [Ca2þ]i elevation was suYcient to induce LTP in rat hippocampal CA1 synapses onto the injected neuron, while a more prolonged but modest [Ca2þ]i elevation specifically induced LTD, and a brief but modest Ca2þ rise could elicit either (Malenka et al., 1988; Neveu and Zucker, 1996a,b; Yang et al., 1999). By terminating the [Ca2þ]i rise following a brief aVerent tetanus by photoactivating the Ca2þ chelator diazo-4, we showed that postsynaptic [Ca2þ]i must remain elevated for several seconds before it can induce (Malenka et al., 1992). We also found that long-lasting changes in synaptic transmission at CA3 hippocampal pyramidal cells can be produced by postsynaptic [Ca2þ]i elevations induced by DM-nitrophen, NP-EGTA, or DMNPE-4 photolysis (Wang et al., 2004). A diVerent form of LTD in cerebellar Purkinje neurons that plays a role in motor skill learning, parallel fiber synapses are depressed when their activity coincides with postsynaptic firing, especially when the latter is triggered by climbing fiber input. Lev-Ram et al. (1997) showed that photolytic release of caged Ca2þ from nitr-7 could replace postsynaptic spiking and that photolytic release of either caged NO or caged cGMP could replace parallel fiber activity; simultaneous uncaging of Ca2þ and either NO or cGMP could induce LTD without any electrical stimulation at all. Kasono and Hirano (1994) showed that a modest release of Ca2þ from nitr-5 depressed responses to glutamate application to a dendrite only when the stimuli were temporally paired. Using DMNPE-4, Tanaka et al. (2007) found that a suYciently high and prolonged Ca2þ elevation alone could induce LTD, and that the threshold for LTD induction was historydependent. Depolarization-induced suppression of inhibition (DSI) is another form of cortical synaptic plasticity, which is mediated by activation of postsynaptic endocannabinoid synthesis by activity-induced [Ca2þ]i elevation and subsequent retrograde regulation of inhibitory transmitter release. We found identical DSI
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sensitivities to uniform postsynaptic [Ca2þ]i elevation by NP-EGTA photolysis vs. the volume-average of the highly nonuniform [Ca2þ]i elevation on opening voltagesensitive Ca2þ channels by depolarizations (Wang and Zucker, 2001), implying that the enzymatic targets of postsynaptic Ca2þ entering through Ca2þ channels in activating DSI are not tightly colocalized with the channels—a situation exactly opposite of the case for Ca2þ activation of classical transmitter release. Long-lasting synaptic regulation also occurs at developing neuromuscular junctions, where repeated activation of one of two motor neuron inputs results in a postsynaptic Ca2þ-dependent compensatory or homeostatic reduction in presynaptic transmitter release to action potentials at terminals facing the activated receptors. Using focal DM-nitrophen or nitr-5 photolysis to mimic the localized postsynaptic [Ca2þ]i elevation seen to accompany the stimulus normally used to induce this selective persistent depression, we were able to induce a similar synapse-specific modification (Cash et al., 1996a). Subsequently, synapses made by the modified motor neuron onto other muscle fibers also became depressed by the spread of an unidentified presynaptic intracellular signal (Cash et al., 1996b). D. Other Applications The tight regulation of cytoplasmic [Ca2þ]i is essential for ensuring that Ca2þ can act reliably and eYciently as a localized second messenger of a huge variety of cellular processes. Endogenous buVers play a defining role in this process, and an appreciation of the functions of these buVers and their characteristics (aYnities, binding kinetics, mobility, and localization) is crucial to our understanding how Ca2þ performs its central cellular functions. Use of photosensitive Ca2þ chelators has become an important tool in the estimation of cytoplasmic buVer characteristics, and much eVort has gone into developing procedures and protocols for defining them with some precision. Some of the best examples of this sort of analysis come from the laboratories of Stephen Bolsover, Istvan Mody and Julio Vergara, and Erwin Neher, whose papers should be consulted for the analytical details (Faas et al., 2007; Fleet et al., 1998; Na¨gerl et al., 2000; Naraghi et al., 1998; Xu et al., 1997). In addition to these major areas of application of caged Ca2þ chelators, this method of [Ca2þ]i manipulation has been used to address an increasingly diverse range of biological problems. Nitr and diazo compounds were inserted by AM loading into fibroblasts that were activated by mitogenic stimulation to produce [Ca2þ]i oscillations monitored using f1uo-3 (Harootunian et al., 1988). Photorelease of Ca2þ from nitr-5 enhanced and accelerated the oscillations, whereas release of caged chelator by photolysis of diazo-2 inhibited them. Nitr-7 photolysis caused not only an immediate rise in [Ca2þ]i liberated from the photolyzed chelator, but also elicited a later rise in [Ca2þ]i (Harootunian et al., 1991). This eVect was shown, pharmacologically, to be caused by IP3-sensitive stores, suggesting that an interaction between [Ca2þ]i and these stores underlies the [Ca2þ]i oscillations. Photorelease of Ca2þ from DM-nitrophen has been used to study the binding kinetics of Ca2þ to the Ca2þ-ATPase of sarcoplasmic reticulum vesicles (DeLong
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et al., 1990). The relaxation of the [Ca2þ] step, measured by arsenazo spectrophotometry after photolysis, revealed the kinetics of binding to the ATPase. Changes in the Fourier transform infrared spectrum consequent to photorelease of Ca2þ from nitr-5 provided information on structural changes in the ATPase after binding Ca2þ (Buchet et al., 1991, 1992). In a final application to the study of enzyme conformational changes, photolysis of Mg2þ-loaded DM-nitrophen was used to form Mg2þ-ATP rapidly to activate Naþ/Kþ exchange, the state of which was monitored by fluorescence of aminostyrylpyridinium dyes (Forbush and Klodos, 1991). Rate-limiting steps were measured at 45 s 1 by this method. Ca2þ has been implicated in the control of filopodial activity in the responses of growth cones of developing neurons to environmental cues. Pioneer neurons lay out peripheral aVerent pathways in developing grasshoppers. We loaded pioneer neurons by de-esterification of the AM esters of DM-nitrophen and calcium green (Lau et al., 1999) and showed that elevation of local [Ca2þ]i in a growth cone to 1 mM for just 10 s was suYcient to activate subsequent filopodial prolongation and induce the formation of new filopodia at spots with high actin concentration (labeled with rhodamine-phalloidin). In other applications, Gilroy et al. (1991) and Fricker et al. (1991) microinjected Ca2þ-loaded nitr-5 into guard cells of lily leaves and showed that photorelease of about 600 nM intracellular Ca2þ (measured with fluo-3) initiated stomatal pore closure. Kao et al. (1990) loaded Swiss 3T3 fibroblasts with nitr-5/AM and showed that photolysis that elevated [Ca2þ]i by hundreds of nanomolar (measured by fluo-3) triggered nuclear envelope breakdown, an early step in mitosis, while having little eVect on the metaphase to anaphase transition. Control experiments using nitr-9 showed no eVect of reactive photochemical intermediates or products. Groigno and Whitaker (1998) initiated chromosome disjunction and segregation in embryonic sea urchin cells by Ca2þ photorelease from NP-EGTA or by photolysis of caged IP3. Ca2þ buffers prevented chromatid separation but not the later stages of anaphase, indicating a specific role for Ca2þ in early anaphase chromosome disjunction. Tisa and Adler (1992) used electroporation to introduce Ca2þloaded nitr-5 or DM-nitrophen into Escherichia coli bacteria, and showed that elevation of [Ca2þ]i enhanced tumbling behavior characteristic of chemotaxis whereas photorelease of caged chelator from diazo-2 decreased tumbling. Photolysis of diazo-3, which reduces pH without aVecting [Ca2þ]i, caused only a small increase in tumbling. Mutants with methyl-accepting chemotaxis receptor proteins still responded to Ca2þ, whereas mutants of specific Che proteins did not, indicating that the action of these proteins lay downstream of the Ca2þ signal.
X. Conclusions Interest in photolabile Ca2þ chelators has been intense. Their range of application has broadened well beyond the original nerve, muscle, and fibroblast preparations. They remain one of the most valuable tools for the precise definition of
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calcium’s roles and mechanisms of action in cell biology. They have attracted and challenged some of the best minds in physiology, resulting in great conceptual and skillful sophistication in the rapid evolution of this technology, which shows little sign of abating. Acknowledgments I thank Steve Adams for valuable discussion and Joseph Kao for drawings of chelator structures. The research done in my laboratory in this area was supported primarily by National Institutes of Health Grant NS 15114.
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Robert Zucker Buchet, R., Jona, I., and Martonosi, A. (1992). The eVect of dicyclohexylcarbodiimide and cyclopiazonic acid on the diVerence FTIR spectra of sarcoplasmic reticulum induced by photolysis of cagedATP and caged-Ca2þ. Biochim. Biophys. Acta 1104, 207–214. Carl, A., McHale, N. G., Publicover, N. G., and Sanders, K. M. (1990). Participation of Ca2þ-activated Kþ channels in electrical activity of canine gastric smooth muscle. J. Physiol. (Lond.) 429, 205–221. Cash, S., Dan, Y., Poo, M.-M., and Zucker, R. (1996). Postsynaptic elevation of calcium induces persistent depression of developing neuromuscular synapses. Neuron 16, 745–754. Cash, S., Zucker, R. S., and Poo, M.-M. (1996). Spread of synaptic depression mediated by presynaptic cytoplasmic signaling. Science 272, 998–1001. Charnet, P., Richard, S., Gurney, A. M., Ouadid, H., Tiaho, F., and Nargeot, J. (1991). Modulation of Ca currents in isolated frog atrial cells studied with photosensitive probes. Regulation by cAMP and Ca2þ: A common pathway? J. Mol. Cell. Cardiol. 23, 343–356. Delaney, K. R., and Zucker, R. S. (1990). Calcium released by photolysis of DM-nitrophen stimulates transmitter release at squid giant synapse. J. Physiol. (Lond.) 426, 473–498. DeLong, L. J., Phillips, C. M., Kaplan, J. H., Scarpa, A., and Blasie, J. K. (1990). A new method for monitoring the kinetics of calcium binding to the sarcoplasmic reticulum Ca2þ-ATPase employing the flash-photolysis of caged-calcium. J. Biochem. Biophys. Methods 21, 333–339. DelPrincipe, F., Egger, M., Ellis-Davies, G. C., and Niggli, E. (1999). Two-photon and UV-laser flash photolysis of the Ca2þ cage, dimethoxynitrophenyl-EGTA-4. Cell Calcium 25, 85–91. Denk, W. (1997). Pulsing mercury arc lamps for uncaging and fast imaging. J. Neurosci. Methods 72, 39–42. Denk, W., Strickler, J. H., and Webb, W. W. (1990). Two-photon laser scanning fluorescence microscopy. Science 248, 73–76. Duncan, G., Rabl, K., Gemp, I., Heidelberger, R., and Thoreson, W. B. (2010). Quantitative analysis of synaptic release at the photoreceptor synapse. Biophys. J. 98, 2102–2110. Eberhard, M., and Erne, P. (1991). Calcium binding to fluorescent calcium indicators: Calcium green, calcium orange and calcium crimson. Biochem. Biophys. Res. Commun. 180, 209–215. Eberius, C., and Schild, D. (2001). Local photolysis using tapered quartz fibres. Pflu¨gers Arch. 443, 323–330. Ellis-Davies, G. C. (2003). Development and application of caged calcium. Methods Enzymol. 360, 226–238. Ellis-Davies, G. C. (2006). DM-nitrophen AM is caged magnesium. Cell Calcium 39, 471–473. Ellis-Davies, G. C., and Barsotti, R. J. (2006). Tuning caged calcium: Photolabile analogues of EGTA with improved optical and chelation properties. Cell Calcium 39, 75–83. Ellis-Davies, G. C. R., and Kaplan, J. H. (1988). A new class of photolabile chelators for the rapid release of divalent cations: Generation of caged Ca and caged Mg. J. Org. Chem. 53, 1966–1969. Ellis-Davies, G. C., and Kaplan, J. H. (1994). Nitrophenyl-EGTA, a photolabile chelator that selectively binds Ca2þ with high aYnity and releases it rapidly upon photolysis. Proc. Natl. Acad. Sci. USA 91, 187–191. Ellis-Davies, G. C., Kaplan, J. H., and Barsotti, R. J. (1996). Laser photolysis of caged calcium: Rates of calcium release by nitrophenyl-EGTA and DM-nitrophen. Biophys. J. 70, 1006–1016. Engert, F., Paulus, G. G., and BonhoeVer, T. (1996). A low-cost UV laser for flash photolysis of caged compounds. J. Neurosci. Methods 66, 47–54. Escobar, A. L., Cifuentes, F., and Vergara, J. L. (1995). Detection of Ca2þ-transients elicited by flash photolysis of DM-nitrophen with a fast calcium indicator. FEBS Lett. 364, 335–338. Escobar, A. L., Ve´lez, P., Kim, A. M., Cifuentes, F., Fill, M., and Vergara, J. L. (1997). Kinetic properties of DM-nitrophen and calcium indicators: Rapid transient response to flash photolysis. Pflu¨gers Arch. 434, 615–631. Faas, G. C., Karacs, K., Vergara, J. L., and Mody, I. (2005). Kinetic properties of DM-nitrophen binding to calcium and magnesium. Biophys. J. 88, 4421–4433. Faas, G. C., Schwaller, B., Vergara, J. L., and Mody, I. (2007). Resolving the fast kinetics of cooperative binding: Ca2þ buVering by calretinin. PLoS Biol. 5, e311.
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Robert Zucker Hadley, R. W., Kirby, M. S., Lederer, W. J., and Kao, J. P. (1993). Does the use of DM-nitrophen, nitr-5, or diazo-2 interfere with the measurement of indo-1 fluorescence? Biophys. J. 65, 2537–2546. Harootunian, A. T., Kao, J. P., and Tsien, R. Y. (1988). Agonist-induced calcium oscillations in depolarized fibroblasts and their manipulation by photoreleased Ins(1, 4, 5)P3, Caþþ, and Caþþ buVer. Cold Spring Harb. Symp. Quant. Biol. 53(Pt. 2), 935–943. Harootunian, A. T., Kao, J. P., Paranjape, S., Adams, S. R., Potter, B. V., and Tsien, R. Y. (1991). Cytosolic Ca2þ oscillations in REF52 fibroblasts: Ca2þ-stimulated IP3 production or voltage-dependent Ca2þ channels as key positive feedback elements. Cell Calcium 12, 153–164. Haydon, P. G., Man-Son-Hing, H., Doyle, R. T., and Zoran, M. (1991). FMRFamide modulation of secretory machinery underlying presynaptic inhibition of synaptic transmission requires a pertussis toxin-sensitive G-protein. J. Neurosci. 11, 3851–3860. Heidelberger, R., Heinemann, C., Neher, E., and Matthews, G. (1994). Calcium dependence of the rate of exocytosis in a synaptic terminal. Nature 371, 513–515. Heinemann, C., Chow, R. H., Neher, E., and Zucker, R. S. (1994). Kinetics of the secretory response in bovine chromaYn cells following flash photolysis of caged Ca2þ. Biophys. J. 67, 2546–2557. Hochner, B., Parnas, H., and Parnas, I. (1989). Membrane depolarization evokes neurotransmitter release in the absence of calcium entry. Nature 342, 433–435. Hosoi, N., Sakaba, T., and Neher, E. (2007). Quantitative analysis of calcium-dependent vesicle recruitment and its functional role at the calyx of Held synapse. J. Neurosci. 27, 14286–14298. Hsu, S. F., Augustine, G. J., and Jackson, M. B. (1996). Adaptation of Ca2þ-triggered exocytosis in presynaptic terminals. Neuron 17, 501–512. Iino, M., and Endo, M. (1992). Calcium-dependent immediate feedback control of inositol 1,4,5triphosphate-induced Ca2þ release. Nature 360, 76–78. Johnson, B. D., and Byerly, L. (1993). Photo-released intracellular Ca2þ rapidly blocks Ba2þ current in Lymnaea neurons. J. Physiol. (Lond.) 462, 321–347. Kamiya, H., and Zucker, R. S. (1994). Residual Ca2þ and short-term synaptic plasticity. Nature 371, 603–606. Kao, J. P., and Adams, S. R. (1993). Photosensitive caged compounds: Design, properties, and biological applications. In ‘‘Optical Microscopy: New Technologies and Applications,’’ (B. Herman, and J. J. Lemasters, eds.), pp. 27–85. Academic Press, New York. Kao, J. P., Harootunian, A. T., and Tsien, R. Y. (1989). Photochemically generated cytosolic calcium pulses and their detection by fluo-3. J. Biol. Chem. 264, 8179–8184. Kao, J. P., Alderton, J. M., Tsien, R. Y., and Steinhardt, R. A. (1990). Active involvement of Ca2þ in mitotic progression of Swiss 3T3 fibroblasts. J. Cell Biol. 111, 183–196. Kaplan, J. H. (1990). Photochemical manipulation of divalent cation levels. Annu. Rev. Physiol. 52, 897–914. Kaplan, J. H., and Ellis-Davies, G. C. (1988). Photolabile chelators for the rapid photorelease of divalent cations. Proc. Natl. Acad. Sci. USA 85, 6571–6575. Kaplan, J. H., and Somlyo, A. P. (1989). Flash photolysis of caged compounds: New tools for cellular physiology. Trends Neurosci. 12, 54–59. Kaplan, J. H., Forbush, B., 3rd, and HoVman, J. F. (1978). Rapid photolytic release of adenosine 50 triphosphate from a protected analogue: Utilization by the Na:K pump of human red blood cell ghosts. Biochemistry 17, 1929–1935. Kasai, H., Kishimoto, T., Liu, T. T., Miyashita, Y., Podini, P., Grohovaz, F., and Meldolesi, J. (1999). Multiple and diverse forms of regulated exocytosis in wild-type and defective PC12 cells. Proc. Natl. Acad. Sci. USA 96, 945–949. Kasono, K., and Hirano, T. (1994). Critical role of postsynaptic calcium in cerebellar long-term depression. Neuroreport 6, 17–20. Kentish, J. C., Barsotti, R. J., Lea, T. J., Mulligan, I. P., Patel, J. R., and Ferenczi, M. A. (1990). Calcium release from cardiac sarcoplasmic reticulum induced by photorelease of calcium or Ins(1, 4, 5)P3. Am. J. Physiol. 258, H610–H615.
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CHAPTER 3
Making and Using Calcium-Selective Mini- and Microelectrodes L. Hove-Madsen,* S. Baudet,† and D. M. Bers‡ *Cardiovascular Research Centre CSIC-ICCC Hospital de la Santa Creu i Sant Pau Barcelona, Spain †
Ricerca Biosciences SAS Saint Germain sur l’Arbresle, France
‡
Department of Pharmacology University of California, Davis, Davis, California, USA
Abstract I. Introduction A. Main Characteristics of Ca2þ-Selective Electrodes II. Rationale III. Methods A. Preparation of Minielectrodes B. Application of Minielectrodes C. Preparation of Ca2þ-Selective MEs D. Application of Ca2þ-Selective MEs IV. Discussion References
Abstract Detection and measurement of intracellular calcium concentration ([Ca2þ]i) have relied on various methods, the popularity of which depends on their ease of use and applicability to diVerent cell types. Historically, Ca2þ-selective electrodes have been used concomitantly with absorption indicators such as arsenazo-III, but their interest has been eclipsed by the introduction of a large number of fluorescent calcium probes with calcium sensitivities varying from the nanomolar METHODS IN CELL BIOLOGY, VOL. 99 Copyright 2010, Elsevier Inc. All rights reserved.
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to the micromolar range such as fura-2, indo-1, fluo-4, and many others. In this chapter, we emphasize the utility of Ca2þ-selective electrodes and show that their use is complementary to use of fluorescent indicators; indeed, each method has advantages and disadvantages. We first describe the preparation and application of Ca2þ-selective minielectrodes based on the Ca2þ ligand ETH 129 (Schefer et al., 1986) that have a larger dynamic range and faster response time than most commercially available calcium electrodes. The second part of the chapter is dedicated to ETH 129-based Ca2þ-selective microelectrodes (MEs), and their application in the determination of [Ca2þ]i in cardiac cells. Since numerous reviews and books have been dedicated to the theoretical aspects of ion-selective ME principles and technology, this chapter is not intended for investigators who have no experience with MEs.
I. Introduction A. Main Characteristics of Ca2þ-Selective Electrodes The key advantage of the Ca2þ-selective electrodes is the wide dynamic range of their response (e.g., from pCa 9 to 1), as compared, for example, to fluorescent and metallochromic Ca2þ indicators that typically have a dynamic range of four or less pCa units (Fig. 1). There has been developed a plethora of useful fluorescent calcium probes with calcium sensitivities varying from the nanomolar to the micromolar range such as fura-2, indo-1, fura red, fluo-4, furaptra, fluo-5N, and others (Grynkiewicz et al., 1985; Harkins et al., 1993; Lipp et al., 1996; Picht et al., 2006; Shannon and Bers, 1997). These are widely used and are extremely important tools for study of Ca2þ, but Ca2þ-selective electrodes are a valuable complementary tool. For more basic reference to electrode technology and electrophysiology, we suggest monographs by Ammann (1986), Purves (1981), and Thomas (1982). An electronic introduction to ion-selective electrodes can be found at www.nico2000.net/Book/ Guide1.html. Their response is based on a semiempirical equation (Nicolski–Eisenman equation) derived from the Nernst equation: pot zx=zy ay Þ Ex ¼ Eo þ RT =Zx F lnðax þ Kxy
ð1Þ
where Ex is the ion-selective electrode potential, Eo is a constant, R, T, Z, and F have their usual meaning, ax is the activity of the ion that is measured (activity (a) is related to concentration (C) by the relation: a ¼ gC where g is the activity coeYcient) and Kxypot is the selectivity coeYcient. This expression is strictly valid for activities only, but if the activity coeYcients do not change, they can be used with free concentrations too. This is often for convenience, since solutions and chemical equilibria are more often described in these concentrations terms. So, if x is Ca2þ and y is Naþ (the most common interfering cation
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100
3
75
Indo-1
2
Potential (mV)
50 1 25 0 0
Fluorescence ratio
3. Calcium Selective Mini- and Microelectrodes
Minielectrode Microelectrode
−25 −50 −75
5 4 3 pCa Fig. 1 Dynamic range of Ca2þ-selective electrodes and indo-1. The electrode potential of Ca2þselective mini and MEs is shown together with the fluorescence ratio (400/470) for indo-1. Measurements were performed in a KCl buVer containing 140 mM KCl, 10 mM HEPES, 10 mM NaCl, and 1 mM EGTA. Notice that indo-1 is suitable for measurements between pCa 7.5 and 5, while the dynamic range for the Ca2þ electrode is wider, ranging from pCa 9 to 1 for minielectrodes and 7.5 to 1 for MEs. 8
7
6
for the Ca2þ-selective ligand), the relationship becomes, for 30 C and changing to log 10: 2 pot ½Naþ ð2Þ ECa ¼ Eo þ 30 log Ca2þ þ KNaCa Thus for the case of no interfering ion and when extracellular Ca2þ ¼ [Ca2þ]ref, then the potential diVerence (DE) between two solutions of diVerent [Ca2þ] reduces to the Nernst equation: DE ¼ 30 logð½Ca2þ =½Ca2þ ref Þ
ð3Þ
The response of an ion-selective electrode to changes in free [Ca2þ] is much slower than the fluorescent, bioluminescent, and metallochromic Ca2þ indicators. Thus, Ca2þ electrodes are ideal for measurements of slow changes over wide ranges of [Ca2þ], but not appropriate for very rapid changes of free [Ca2þ] although the Ca2þ electrodes can respond in the millisecond range at higher [Ca2þ] (Bers, 1983). The response time can also be improved by using Ca2þ selective electrodes with a concentric inner micropipette that reduces the longitudinal resistance of the Ca2þ-selective resin and thereby decreases the electrical time constant (Fedirko et al., 2006; Ujec et al., 1979). These limitations, together with the physical size of Ca2þ-selective electrodes put them at disadvantage with fluorescent and luminescent calcium indicators for measurements of dynamic changes in calcium levels in
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cellular environments. Moreover, about the time that indo-1 and fura-2 were synthesized (Grynkiewicz et al., 1985), a Ca2þ-ligand of improved selectivity (ETH 129) was introduced (Schefer et al., 1986), which made it more realistic to measure low Ca2þ levels typical of intracellular environments, since the electrode response was Nernstian until pCa 8–9 (depending on the ionic background). However, the availability and popularity of the fluorescent indicators did to a large extent eclipse and limit further use and characterization of ETH 129-based electrodes.
II. Rationale We will not present an extensive review of the advantages and disadvantages of Ca2þ-selective electrodes compared to fluorescent indicators, but the purpose of this chapter is rather to show how the advantages of both approaches can be combined for specific purposes. For example, a classical problem with Ca2þ indicators is to convert the fluorescent signal to actual [Ca2þ]. In fact, it is commonly acknowledged that in vitro calibration curves of fluorescent calcium indicators are not applicable to in vivo or in situ conditions, because the indicators bind to intracellular constituents, which modifies their excitation/emission spectrum and decreases the apparent aYnity of Ca2þ for the probe (Blatter and Wier, 1992; Harkins et al., 1993; Hove-Madsen and Bers, 1992; Konishi et al., 1988). In vivo calibration curves are even more diYcult to obtain and are highly dependent on the cell type under study. Ca2þ-selective electrodes are still one of the most straightforward ways to measure and quantify Ca2þ because (1) their behavior is not appreciably altered by the intracellular milieu (ions and proteins); (2) they can be easily included in an electrophysiological setup and do not require extensive and expensive apparatus; (3) their linear (Nernstian) response simplifies the conversion of the voltage signal to [Ca2þ]i; (4) their behavior allows determination of wide ranges of pCa (see above); and (5) the Ca2þ ligand itself does not change (or buVer) [Ca2þ]i. Thus, Ca2þ-selective electrodes continue to be a good choice for the preparation of calibration solutions for Ca2þ determinations and for measurements of dissociation constants for Ca2þ-binding compounds such as ethylene glycol bis(b-amino ethyl ether)-N,N,N0 ,N0 -tetraacetic acid (EGTA), indo-1, 1,2-bis(o-aminophenoxy) ethane-N,N,N0 ,N0 -tetraacetic acid (BAPTA), and oxalate under diVerent experimental conditions (Bers, 1982; Harrison and Bers, 1987, 1989; Hove-Madsen and Bers, 1992, 1993a; Hove-Madsen et al., 1998) Indeed, commercially available Ca2þ electrodes are largely directed towards determination of the calcium concentration in solutions or biological fluids, but Ca2þ electrodes can also be used to measure cellular Ca2þ buVering and changes in the free [Ca2þ] in cell suspensions (HoveMadsen and Bers, 1993a, 1993b; Hove-Madsen et al., 1998) or combined with other electrophysiological techniques (Kang and Hilgemann, 2004; Kang et al., 2003). Moreover, Ca2þ electrodes are economical, easy to prepare, and they can
3. Calcium Selective Mini- and Microelectrodes
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easily be used for measurements of free [Ca2þ] in experimental solutions or biological fluids. Indeed, for those studying Ca2þ-dependent processes, there is no practical or economical reason why Ca2þ electrodes should not be used as routinely as pH electrodes.
III. Methods A. Preparation of Minielectrodes Ca2þ-selective minielectrodes can be prepared by dipping polyethylene (PE) tubes (typically 5 cm) in a membrane solution (see composition below). We have tried other types of tubing but polyvinyl chloride (PVC) tubing appears to absorb ETH 129 from the Ca2þ-selective membrane, resulting in a faster loss of sensitivity as compared to the PE tubing. On the other hand, materials such as Teflon tubing absorb little ETH 129 but the PVC membrane does not adhere well to the tubing. As a result, the electrodes are more easily damaged, although they may have a longer lifetime if handled with care. The dimensions of the tubing vary from a diameter less than 1 to 3 mm. Electrodes prepared with the membrane solution described below result in Ca2þselective membranes that are a few hundred micrometers thick. With diameters larger than 5 mm, the Ca2þ-selective membrane bursts more easily during handling, but this problem may be overcome by inserting a ceramic plug into the tubing, before dipping it in the membrane solution as described by Orchard et al. (1991). For general purposes we have used inner electrode diameters of 1.67 mm (PE 240, Clay Adams). After dipping the PE tubing in the membrane solution, the Ca2þ-selective membrane is allowed to dry overnight. Then the electrode is filled with an appropriate filling solution, which should correspond to experimental conditions (see below). After filling the electrode, it is allowed to equilibrate for at least 3 days in a glass vial containing the filling solution (but see below).
1. Preparation and Use of the Ca2þ-Selective Ligand The Ca2þ-selective membrane can be prepared as described by Schefer et al. (1986) (all from Fluka/Sigma-Aldrich; Table I). ETH 129 is dissolved in N-phenyl-octyl-ether (NPOE) under vigorous stirring in a small glass vial (Solution 1). At the same time, PVC is dissolved in the THF; when completely dissolved, potassium TCPB is added (Solution 2). When the components of Solutions 1 and 2 are completely dissolved, the two solutions are mixed, and the membrane solution is ready to use, or can be stored in a glass vial, closed with a Teflon screwcap and protected from light. If THF evaporates from the membrane solution during storage, a small amount of THF can be added to achieve the desired viscosity of the membrane solution.
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72 Table I Preparation of minielectrodes
the
PVC-based
Ca2þ-selective
ligand
Component*
Amount
Solution 1 ETH 129 N-phenyl-octyl-ether (NPOE)
25 mg 451.5 ml
Solution 2 Polyvinyl chloride (PVC) Kþ-tetrakis chlorophenyl borate (TCPB) Tetrahydrofuran (THF)
250 mg 12.9 mg 5 ml
*
Components can be obtained from Sigma-Aldrich (St. Louis, MO)
The filling solution used for the minielectrode depends on the experimental solutions. Generally, the ionic composition should mimic the environment in which measurements of Ca2þ are planned, and the [Ca2þ] of the filling solution should also be in the range of the measured values. With measurements of low [Ca2þ], Ca2þ in the filling solution can be buVered with EGTA to the desired free [Ca2þ]. However, we have not obtained good results with filling solutions with a pCa higher than 7.5. We typically use a Ca2þ-EGTA buVer with 1 mM free Ca2þ as a filling solution.
2. Electrode Characteristics The resistance of the minielectrodes is 1–2 MO, which normally makes it possible to use a standard pH/ion meter to monitor the electrode potential. We have used either a commercial pH/ion meter (Orion pH/ion analyzer) or preamplifier (A311J, Analog Devices). When using commercial pH/ion meters, it is normally necessary to make an adapter cable that connects the minielectrode to the meter input. We have used a chart recorder (Soltec) or an electronic data acquisition device (Linseis) for continuous monitoring of the electrode potential. The lifetime of the minielectrode depends on the [Ca2þ] the electrode is used to measure, and on the composition of the experimental solutions. For measurements in solutions without protein or interfering ions, the detection limit for the electrodes increases slowly with time and the electrodes will have a detection limit in the subnanomolar range and a Nernstian response down to 10 nM for at least 1 month after preparation. Figure 2 shows the change in the response of a Ca2þ electrode with time. Two days after filling, the electrode response was still ‘‘super-Nernstian’’ at low [Ca2þ] (i.e., > 29 mV per 10-fold change in [Ca2þ]), but normal within 7 days after filling. Notice that we did still obtain a Nernstian response down to pCa 8 for 2 months after filling the electrode. The response time at low [Ca2þ], however, got slower with
73
3. Calcium Selective Mini- and Microelectrodes
50
Potential (mV)
25 0 −25
2 Months
−50 −75
2 Days
−100 8
7
6 5 4 pCa Fig. 2 Electrode potential of a Ca2þ-selective minielectrode 2, 7 and 60 days after filling of the minielectrode. Measurements were performed in a KCl buVer as in Fig. 1. Notice the ‘‘super-Nernstian’’ response of electrodes 2 days after filling (circles). Seven days after the filling (squares), electrode response was linear down to a free [Ca2þ] of less than 10 nM. The slope of the regression line was 28.4 mV/pCa. Two months after filling (diamonds), the electrode response was linear to pCa 7.5, but response time was slowed at high pCa.
time and measurements below 30 nM are only practical with fairly fresh electrodes. We generally fill electrodes once a week to obtain the best results. However, if the Ca2þ electrodes are used to measure micromolar or higher [Ca2þ] in protein-free solutions, the same electrode can be used for longer periods (up to several months). In the presence of cellular proteins, a small oVset in the electrode response is seen at the first exposure to protein. Then, no further alteration of the electrode response occurs, but the response time of the Ca2þ electrode is increased after exposure to protein, and measurements of free [Ca2þ] below 10 nM are more diYcult in the presence of protein concentrations higher than 10 mg/ml. The response time of the Ca2þ electrodes can be of critical importance in some applications. Figure 3 compares the response times of a Ca2þ electrode and indo-1 fluorescence to a decrease in the [Ca2þ] in a suspension of permeabilized myocytes (3 mg/ml) where cellular Ca2þ uptake processes have been blocked with thapsigargin and ruthenium red. We examined the response to a decrease in [Ca2þ], as this may be a more stringent test than an increase in [Ca2þ]. Notice that when 2 mM EGTA was added to lower the free [Ca2þ], the electrode response was 94% complete in 1 s while the indo-1 signal was 97% complete in 1 s. A slow final phase, lasting several seconds, is apparent in the electrode signal only (see amplified inset). In Fig. 3 the response time was examined under experimental conditions where spatial inhomogeneities in the myocyte suspension are minimized by buVering Ca2þ with indo-1 and oxalate. However, under some experimental conditions, an
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EGTA
Free [Ca] (mM)
40 30
Free [Ca] (mM)
Indo
50
1.5
Elec
1 0.5 0 0
Elec 20
10 Time (s)
10
0
−10
−5
0
5 10 15 Time (s) Fig. 3 Comparison of response time of a Ca2þ-selective minielectrode and indo-1. Free [Ca2þ] was measured in a suspension of permeabilized rabbit ventricular myocytes. Ca2þ uptake into sarcoplasmic reticulum and mitochondria was inhibited with thapsigargin and ruthenium red, respectively. The initial free [Ca2þ] was 32 mM which is near saturation for indo-1, resulting in a very noisy trace. That is because a small change in fluorescence ratio corresponds to a large in [Ca2þ] at this level (see Fig. 1). At the arrow, 2 mM EGTA was added to the cell suspension to lower free [Ca2þ]. Both the electrode and indo-1 signal were more than 90% complete in 1 s. The inset shows the response of indo-1 and Ca2þ electrode at low [Ca2þ]. Notice that the indo-1 signal was 100% complete in 2 s (and actually undershoot slightly) while the final completion of the electrode response was slower.
apparently slower electrode response may result from inhomogeneities rather than a slower electrode response per se. This is illustrated in Fig. 4, where free [Ca2þ] was monitored with Ca2þ electrode and indo-1 simultaneously in a myocyte suspension in the absence and presence of oxalate. In Fig. 4A, Ca2þ addition to the cells causes a rapid increase in [Ca2þ], which is subsequently sequestered by the sarcoplasmic reticulum (SR). In the absence of 10 mM oxalate (Fig. 4A), the electrode response appears to be slower than the corresponding indo-1 signal. However, when oxalate is subsequently added to the cell suspension (Fig. 4B), the measured change in free [Ca2þ] after a Ca2þ addition is similar for indo-1 and the Ca2þ electrode. It should be noted that oxalate not only buVers the free [Ca2þ], but also increases the Ca2þ uptake rate in the SR, and thereby the removal of Ca2þ from the cell suspension. Thus, despite inducing a faster rate of change in free [Ca2þ], oxalate eliminates the diVerence between Ca2þ electrode and indo-1 signal by eliminating spatial inhomogeneities in free [Ca2þ] in the myocyte suspension. Indeed, indo-1 is expected to be less sensitive to spatial inhomogeneities as it diVuses into the permeabilized cells and binds to cellular proteins (Hove-Madsen and Bers, 1992, 1993b). In contrast, the Ca2þ electrode can only measure the Ca2þ outside the permeabilized cells, and inhomogeneities during uptake or release of Ca2þ from the cells are therefore likely to occur, resulting in erroneous measurements with the Ca2þ electrode.
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3. Calcium Selective Mini- and Microelectrodes
2.5
Indo-1
1.5
Ca electrode
1
Free [Ca] (mM)
Free [Ca] (mM)
Indo-1 Ca electrode
15
2
10 5
0.5 0 0 0
250
500 Time (s)
750
1000
0
50
100
150
Time (s)
Fig. 4 EVect of inhomogeneities in [Ca2þ] on a Ca2þ-selective minielectrode and indo-1 response in
permeabilized rabbit ventricular myocytes. Panel A shows simultaneous measurements of Ca2þ uptake in digitonin-permeabilized myocytes with both a Ca2þ-selective minielectrode and indo-1. Ca2þ uptake in mitochondria was inhibited with ruthenium red. Ca2þ was added at time ¼ 0 and was largely accumulated by the SR. Under these conditions, the response of the Ca2þ electrode was slower than indo-1. Panel B shows Ca2þ uptake by precipitating intra-SR Ca2þ and thereby preventing buildup of a [Ca2þ] gradient. Notice that Ca2þ uptake is much faster in the presence of oxalate with no apparent diVerence between electrode and indo-1 response (from Hove-Madsen and Bers (1993a) with permission).
3. Storage of Minielectrodes After PE tubes have been dipped in an ETH 129 membrane solution and allowed to dry overnight, the dry electrodes can be stored in a closed glass vial for long periods. We have filled minielectrodes that had been stored for 3 years and the electrodes made with PE tubing still had a resistance of 1–2 MO with a linear response down to less than 10 nM Ca2þ after filling. Electrodes made with PVC tubing had higher resistance ( 50 MO) but were also functional, although slower and less sensitive. Storage of the electrodes in plastic vials results in ‘‘Ca2þ-selective plastic containers,’’ as the ETH 129 slowly diVuses into the container. Once the Ca2þ electrodes are filled with the filling solution, however, the response time increases and the electrodes gradually lose sensitivity.
B. Application of Minielectrodes Minielectrodes can be used for a number of purposes. The most straightforward application is the preparation of solutions where Ca2þ is buVered with chelators such as EGTA, EDTA, or BAPTA as described by Bers (1982). We have developed a spreadsheet that allows calculation of the actual pCa of these solutions, based on the Nernstian response of the minielectrodes (see Chapter 1). Furthermore, the spreadsheet allows determinations of the Kd and the purity of the Ca2þ chelator used to prepare the solution. Thus we have used the minielectrodes to determine
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the Kd for EGTA, BAPTA, and oxalate in buVer solutions (Bers, 1982; Harrison and Bers, 1987, 1989; Hove-Madsen and Bers, 1993a). More comprehensive programs to calculate the amount of Ca2þ and Ca2þ buVer needed to prepare solutions have been developed (e.g., MaxChelator by C. Patton, Marine Biology Institute, Monterey, CA; http://maxchelator.stanford.edu/downloads.htm; cf. Chapter 1). We have also used the minielectrodes to characterize the binding of Ca2þ to indo-1 in vitro and in cell suspensions, in order to calibrate the indo-1 and furaptra signals when used in cell suspensions (Hove-Madsen and Bers, 1992; HoveMadsen et al., 1998; Shannon and Bers, 1997). In agreement with previous studies of fura-2 (Konishi et al., 1988) we found that indo-1 binds extensively to cellular proteins and causes a fourfold increase in the Kd for Ca2þ-indo-1 in permeabilized myocytes. The minielectrodes can also be used to titrate the passive Ca2þ binding sites in permeabilized myocytes where the cellular Ca2þ uptake and release process are inhibited. Using the same titration method, we have measured total Ca2þ uptake in the SR in permeabilized myocytes, by inhibiting Ca2þ uptake in the mitochondria and release of Ca2þ through the SR Ca2þ release channels (Hove-Madsen and Bers, 1993a). Finally, we have used the minielectrodes together with indo-1 for online measurements of the Ca2þ uptake rate in the SR in permeabilized ventricular myocytes (Hove-Madsen and Bers, 1993b) and to examine the eVects of phospholamban phosphorylation and temperature on the uptake rate (Hove-Madsen et al., 1998; Mattiazzi et al., 1994), and we have determined the inhibition of Ca2þ uptake by thapsigargin in order to measure the number of SR Ca2þ pump sites (HoveMadsen and Bers, 1993b). In experiments measuring Ca2þ uptake rates caution should, however, be taken when using minielectrode because of the above mentioned possibilities of inhomogeneities in Ca2þ in cell suspensions and slowing of the electrode response.
C. Preparation of Ca2þ-Selective MEs
1. Glass Tubing Preparation We have used nonfilamented capillaries (150 mm outer diameter, 15 cm long, from Clark Electromedical Instruments, UK or World Precision Instruments, USA). The glass is cut in the middle with a diamond pen or glass scorer (with care to avoid deposition of dirt). Both ends of the capillaries (now 7.5 cm long) are lightly fire-polished; a whole batch can be prepared and kept in a small glass beaker, preferentially in a dust-proof container. Cleaning of the micropipettes prior to pulling has been a matter of debate, but as 99.8% of the glass after pulling is newly exposed (Deyhimi and Coles, 1982), we do not find such procedures to be necessary.
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2. Microelectrode Pulling and Silanization MEs can be prepared ‘‘on-demand’’ and in ‘‘batch’’ methods. The first method consists in preparing (i.e., pulling and silanizing) MEs on the experimental day, which has the obvious advantage of having ‘‘freshly’’ prepared electrodes and of being able to manufacture as many as desired a day. Protocols have been described (reviewed in Ammann, 1986). The batch method that we use consists of preparing a batch of MEs that can be kept for several days in a dry, dust-proof container. MEs are pulled on a programmable horizontal stage puller (Model P80 PC or comparable, Sutter Instruments, USA). By trial-and-error, and according to the type of biological preparations studied, a satisfactory shape of ME can be found. However, several aspects of ME pulling have to be taken into account when designing its shape. As ion-selective MEs have an intrinsically high resistance (i.e., > 50–100 GO), the signal-to-noise ratio has to be minimized. This can be achieved in two ways: the first, and most obvious, is to increase the tip diameter, within certain limits, which are dictated by the size of the cell type. In our case, we impale cardiac cells in whole muscle. Cell length is typically between 50 and 150 mm and tip diameters less than 0.5 mm are needed. However, there is a trade-oV between tip diameter and detection limit of the electrode (see below). That is, increasing tip diameter improves electrode response (in terms of detection limit and speed of response), but is more likely to damage the cell during impalement. Another simple way to decrease the resistance is to decrease the length of the ME shank. This further helps to reduce capacitative artifacts, that are encountered when the level of physiological solution fluctuates in the experimental bath (Vaughan-Jones and Kaila, 1986), and also helps electrolyte filling (see below). In contracting muscular preparations, the shank should also possess some flexibility to avoid dislodging of the ME during a contraction. Again, by trial-and-error, an adequate shape that fulfills all these requirements can be found. Their shape was designed to reduce their resistance by making them steeply tapered (having a shank length of approximately 150 mm and diameter of 20 mm at 10 mm above the tip). Under light microscopical observation, the tip diameter was estimated to be 0.5 mm. MEs are dehydrated, tip up in an aluminium block, at 200 C for 12 h. Our experience, and of others (Vaughan-Jones and Wu, 1990), has been that a better silanization and longer lifetime of the MEs are achieved this way. The silanization protocol consists of spritzing 300 ml of N,N-dimethyltrimethylsilylamine (Fluka) onto the aluminum block and rapidly placing a glass lid on top of the dish. Care must be taken not to inhale vapor from the silane vial or during its introduction in the dish. In our hands, once opened, the silane vial can be kept for at least 2 months without losing its properties. Silanization procedure lasts for 90 min. The lid is then removed and the MEs are baked for another 60 min, which drives oV the excess silane vapor. The aluminum block and the MEs are then placed into an air-tight plastic container, also containing desiccant. We advise not to keep the MEs more than a week, because repetitive openings of the container and insuYcient seal quality will progressively make the electrodes lose their hydrophobicity.
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3. Electrolyte Filling of the ME The filling or conducting electrolyte is introduced in the ME from the back (back-filling), but because of the hydrophobicity of the glass wall (due to the silanization), the tip does not get filled immediately. The strategy to fill the ME completely depends on the absence or presence of the ligand at its very tip. In the case where the ME is first filled with the electrolyte, the back of the ME is connected, via a flexible tubing, to a 50-ml syringe, whose plunger has been previously pulled to 5 ml. Positive pressure is then applied and, as assessed by microscopic observation, the electrolyte creeps along the ME wall and fills the tip. The biggest air bubbles are then removed by gently flicking the ME, held tip down. Although we have initially applied this procedure to filamented MEs, a similar success rate (more than 90%) has been obtained with nonfilamented MEs (surprising, because silanization is expected to limit aqueous filling ease). Whatever the method used, if the electrolyte column was interrupted by air bubbles then, gentle heating of the tip of the ME, under microscopic control, with a tungsten–platinum wire, according to the device described by Thomas (1982), can remedy this problem. Once filled with electrolyte to the tip, the hydrophobic ligand can be introduced (see below). In the case where the electrolyte is added after the Ca2þ ligand, additional problems exist. In fact, caution has to be taken to avoid the presence of air at the electrolyte–ligand interface. If a traditional whisker is used, great care has to be taken not to accidentally disrupt the column of ligand, which can lead to mixing of oil and water, making unstable ion-selective MEs. If a heating filament is used, care has to be taken not to heat the ligand because it is likely that the local high temperature may damage the ligand properties. We have used a filling solution that has an ionic composition mimicking the intracellular medium (in mM): Naþ: 10; Kþ: 140; HEPES: 10; EGTA: 1; pH 7.1 (at 30 C) and pCa 7. This solution is in fact identical to the calibrating solution having the same pCa (see below and also Orchard et al. (1991)) for additional comments). Our experience has been that it is best to minimize the time between electrolyte and ligand filling. We prefer to fill the ME with the ligand as soon as the ionselective ME is filled with the electrolyte (although we managed to draw the ligand in the tip 2–3 h after electrolyte filling). If MEs are left overnight with the filling solution, it can be very diYcult to draw ligand into the tip; this observation could be explained by a progressive glass hydration, causing it to lose its capability to retain the ligand.
4. Preparation and Use of the Ca2þ-Selective Ligand For repetitive and long-lasting Ca2þ measurements with Ca2þ-selective MEs dissolving the Ca2þ ligand in a ‘‘cocktail’’ containing PVC is useful (Tsien and Rink, 1981). Although the cocktail available from Fluka (Cocktail II containing
79
3. Calcium Selective Mini- and Microelectrodes
Table II Preparation of the PVC-based Ca2þ-selective ligand MEs Componenta
Amount
Solution 1 ETH 129 (or ETH 1001) NPOE Naþ-tetraphenyl borate
27.5 mg 500 ml 5.53 mg
Solution 2 Solution 1 PVC THF
200 ml 36 mg 400 ml
a Components can be obtained from Sigma-Aldrich (St. Louis, MO) and see Table I for abbreviations.
94% (w/w) NPOE, 5% (w/w) ETH 129, and 1% (w/w) sodium TPB) is satisfactory to start with (Ammann et al., 1987), small volumes sometimes provided (0.1 ml) do not facilitate handling. We prefer to make up our own cocktail, in a larger NPOE volume, with the same proportions. The composition, for 500 ml of NPOE is given in Table II. Because of the small volumes and required stirring, it is preferable to work with flat-bottomed, small volume glass vials, and miniature stirring bars. ETH 129 and TPB are dissolved with vigorous stirring in NPOE, in a 2-ml glass vial (Solution 1). Solution 1 can be kept at room temperature for several months, with a Teflon screw cap, protected from light. When the final cocktail (Solution 2) is prepared, PVC is dissolved in THF with stirring. 0.2 ml of Solution 1 is then added, stirred, and finally sonicated. THF is allowed to partially evaporate to approximately half of the initial volume and the final cocktail is finally poured in a 0.5-ml conical vial (Clark Electromedical Instruments). Before dipping the ME tip, THF is allowed to evaporate until the mixture has the consistency of a thick syrup. Experience helps to determine the adequate consistency of the ligand. In fact, if not enough THF has evaporated, the ETH 129 sensor is too diluted and the evaporation in the ME may cause retraction of the gel, yielding poor responses. On the other hand, in some instances, we have managed to fill the electrodes even if the ligand appeared to be solidified (as a rule, the thickest mixture which will fill the tip is best). Because of the small diameter of the tip and the viscosity of the ligand, negative pressure is required at the back of the electrode. This is achieved by a > 10-ml syringe connected, via a 3-way stopcock, to a flexible Teflon tubing connected to the back of the electrode with soft tubing. Vacuum is then applied by pulling the plunger out and by blocking it with a rod/block or collar placed along the plunger (care should be taken to regularly check the vacuum of the system). Observation of the ME and measurements of the ligand column height are performed under
L. Hove-Madsen et al.
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microscopic control, with a microforge (e.g., as described by Thomas (1982). In brief, the microscope body is laid on its back so that the stage (removed) would be vertical and the eyepieces are oriented upwards. A long-working distance objective (40) is used and the ME and the ligand vial are held independently by two micromanipulators. Depending on glass, shape, and tip diameter 10–30 min of negative pressure is typically required to fill the tip. Vacuum is slowly released before lifting the electrode from the ligand. A column height of less than 300 mm is preferable, because it decreases the electrode sensitivity to changes in temperature and level of the bath in the experimental chamber (Vaughan-Jones and Kaila, 1986). Our experience is that, depending on the tip diameter, column heights between 50 and 250 mm yielded acceptable electrode responses. Once the electrode is filled with both the ligand and the electrolyte, we prefer not to let the ME equilibrate in Tyrode or high pCa solutions, because this favors the deposition of dirt on the tip of the ME and might contribute to clogging. Rather, we place them, tip up, in a drilled plastic plate, protected by an upside down glass beaker. Before calibration, the column height is rechecked because THF in the column continues to evaporate, leading to shrinkage of the PVC gel. As THF evaporates, the stock solution becomes even thicker. Periodically enough THF must be added to decrease the viscosity of the mixture. This process is hastened by mixing it with a glass rod and then on a Vortex mixer (maximal setting). Sonication can also be used, but does not give better results.
5. Double-Barreled Ca2þ-Selective MEs For Ca2þ-selective MEs measurements, one must measure both the potential of the Ca2þ electrode and the local voltage (typically with a KCl-filled ME; see below). Both electrodes can be built into a single double-barreled electrode, where one barrel is the Ca2þ electrode and the other is the voltage electrode. We have not had much luck using these for intracellular recording, but they can be extremely useful for measurement of local extracellular or interstitial [Ca2þ] in multicellular preparations (Bers, 1983, 1985, 1987; Bers and MacLeod, 1986; Shattock and Bers, 1989). Double-barreled electrodes can be pulled from 2 to 2.5 mm diameter theta-style tubing (R and D Optical Systems, MD) on a Brown–Flaming P-77 micropipette puller (Sutter Instruments, CA, USA). For extracellular recording, the tips of the electrodes are carefully broken under microscopic observation to have 4–12 mm overall tip diameters. Two methods of silanization of the Ca2þ barrel are practical. (1) Distilled water is injected into the reference barrel. A hypodermic needle containing tri-n-butylchlorosilane is introduced into the Ca2þ barrel, 1 ml of silane is displaced into the shank of each electrode, and electrodes are placed in a 200 C oven, tips up, for 5 min and then cooled. (2) a stream of silanizing N, N-dimethyltrimethylsilylamine (TMSDMA vapor is passed through the Ca2þ barrel (with or without warming), while a stream of nitrogen gas is passed through
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the reference barrel (under pressure) to prevent silanization of the reference barrel (which would result in both barrels being Ca2þ-sensitive). The larger tips of these electrodes make the filling easy. Both barrels can be easily backfilled. The silanized barrel is backfilled with a reference solution containing 10 mM CaCl2 and 100 mM KCl and the nonsilanized barrel with a solution containing 140 mM NaCl. A column of the neutral Ca2þ ion-exchange cocktail ETH 1001 or 129 (Fluka Chemical, Ronkonkoma, NY) 50–250 mm long is easily drawn into the silanized barrel. Ca2þ electrodes with these tip diameters exhibit Nernstian behavior over the range 10 mM–10 mM Ca2þ (Bers and Ellis, 1982). The resistance of these MEs was typically 1–5 GO for the Ca2þ-sensitive barrel and 1–4 MO for the reference barrel. The impedance of the two barrels is very diVerent, but their fast response allows relatively rapid interstitial [Ca2þ] monitoring. To match signal response kinetics to voltage steps a variable-passive R-C filter can be added to the reference barrel signal after the signal has come from operational and oVset amplifiers. This filter is adjusted while a square voltage pulse is fed into the bath until the best matching with the Ca2þ barrel response is obtained (Bers, 1983). These Ca2þ electrodes typically exhibit Nernstian behavior at least over the range 10 mM–10 mM Ca2þ. This is satisfactory for typical extracellular [Ca2þ] measurements and the doublebarreled electrodes are easier to calibrate and use than intracellular impalements (described in more detail below).
6. Calibrating Bath and Solution Perfusion It is preferable to calibrate ion-selective MEs in the experimental chamber in which measurements are made or to have the calibrating bath as close as possible in design and proximity. Our calibration chamber is a ‘‘flow-through’’ type (volume: 0.1 ml), immediately adjacent to the experimental chamber. Note that it is convenient that the experimental chamber is viewed from the front, and not from above.
7. Calibration Procedure The bath electrode is either an Ag wire (chlorided by dipping it in bleach for 15–20 min) or an agar bridge. Ideally, a conventional electrode (3 M KCl filled) should also be immersed in the bath, and the diVerential voltage (ion-selective ME minus conventional) should be read. We use a commercial amplifier (FD-223 from WPI) or a home built amplifier using varactor bridge preamplifiers (AD311J, Analog Devices) as described by Thomas (1982). We have adopted the following method to quickly select suitable MEs. The ME is mounted in its holder and advanced into the calibrating bath, allowing the trace to stabilize. If the device used to measure the signal has a resistance measurement feature, it is worth measuring this parameter. In fact, our experience has been that for the sharp Ca2þ MEs having resistances ranging between 100 and 250 GO were suitable for our experiments, in terms of linearity and detection limit of the
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calibration curve. Within this range, the higher the resistance, the lower the detection limit for a given batch of MEs. At resistances higher than 300 GO, the detection limit decreased sharply, probably because of the small tip diameter (Ammann, 1986). Finally, although low resistance MEs tended to also have low detection limits, they were not suitable for our experiments, because of the large tip diameter that could seriously damage the cell membrane during impalement. By contrast, we have found the height of the ligand column not to be a valuable predictor of ME performance (although this height was always kept 50–250 mm). In our hands, an ME can be used a few minutes after ligand filling. After equilibration in the control physiological solution (in our case, an HEPESbased Tyrode, containing 2 mM Ca2þ), ion-selective ME potential is adjusted to 0 mV. Our 2 mM Ca2þ Tyrode gives a voltage reading corresponding to an intracellular calibrating solution of pCa 2.6. Then, flow is switched to a solution of high pCa (between 7.5 and 9). At 30 C (our experimental temperature), the theoretical slope of the relationship between voltage and pCa is 30 mV/pCa, so that, between pCa 2.6 and pCa 8, the theoretical voltage should be 162 mV. However, as the electrode detection limit decreases at high pCa, a practical compromise is often necessary for acceptability (e.g., readings more negative than 150 mV). A Ca2þ-selective ME meeting this criteria may then be calibrated over a wider range of [Ca2þ]. After the calibration is completed, the ME is moved into the experimental chamber and equilibrated until stable. Conventional, 3 M KCl-filled MEs are pulled from the same glass and with the same characteristics as the Ca2þ electrodes, but are not silanized.
D. Application of Ca2þ-Selective MEs
1. Sharp Ca2þMEs for [Ca2þ]i Measurement We and others have had some, but limited success in making reliable [Ca2þ]i measurements in cardiac myocytes with these electrodes (Bers and Ellis, 1982; Marban et al., 1980), and a few other groups have had some luck with other cell types such as skeletal muscle fibers (Allen et al., 1992; Blatter and Blinks, 1991; Lopez et al., 2000), Aplysia neurones (Gorman et al., 1984), and photoreceptors (Levy and Fein, 1985) using an earlier developed Ca2þ ionophore (ETH 1001; Sigma-Aldrich 21192) instead of ETH 129 in the cocktail. ETH 1001-based MEs generally do not make electrodes that have quite as low a detection limit as ETH 129 (Schefer et al., 1986), but for some reason ETH1001 seems to be of greater practical utility for intracellular Ca2þ MEs. We have done some preliminary cardiac muscle experiments that are consistent with this notion (resting pCa ¼ 6.34 0.15; mean SD; n ¼ 10 determinations). However, with the excellent fluorescent Ca2þ indicators available now that are easy to use, one would need a compelling reason to tackle this challenging electrophysiological approach for [Ca2þ]i and the references above should then help.
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[Ca]o mM
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550 500 480
520
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Fig. 5 Measurements of [Ca2þ]o with double-barreled Ca2þ-selective MEs during single steady state
contractions in (A) rabbit and (B) rat ventricular muscle (0.5 Hz, 30 C). The [Ca2þ]o and tension are shown in the absence and presence of 10 mM citrate (which limits [Ca2þ]o depletion by buVering [Ca2þ]o. Bath [Ca2þ]o ¼ 0.5 mM (dotted line). Data was from Shattock & Bers, (1989), as presented in Bers (2001) (with permission).
2. Measuring Extracellular [Ca2þ] with Double-Barreled MEs Double-barreled Ca2þ MEs can record rapid changes in extracellular [Ca2þ] ([Ca2þ]o) between cells in multicellular preparations such as isolated cardiac trabeculae (Bers, 1983, 1985, 1987; Bers and MacLeod, 1986; Shattock and Bers, 1989). Figure 5A shows that one can detect small [Ca2þ]o depletions during individual steady state rabbit cardiac action potentials and contractions. Moreover, when [Ca2þ]o is buVered by the low aYnity fast buVer citrate these depletions can be suppressed. Note that these [Ca2þ]o depletions reflect net cellular Ca2þ influx (in excess of eZux) early in the contraction and net Ca2þ eZux later in the contraction, such that [Ca2þ]o returns to the bath level. In cardiac myocytes the depletion is driven mainly by Ca2þ influx via Ca2þ channel current and to some extent by Naþ/Ca2þ exchange (which can mediate Ca2þ influx at positive Em when [Ca2þ]i is low). As [Ca2þ]i rises in the cell during the heartbeat because of Ca2þ entry and SR Ca2þ release, it causes enhanced Ca2þ eZux (mainly via Naþ/Ca2þ exchange in cardiac myocytes), and this allows [Ca2þ]o to recover. Note that action potential repolarization greatly enhances the driving force for Ca2þ eZux via Naþ/ Ca2þ exchange, further enhancing the recovery of [Ca2þ]o to the bath level. In rat ventricular muscle the [Ca2þ]o signals are remarkably diVerent (Fig. 5B). In the rat there is only a very brief phase of [Ca2þ]o depletion (for 20 ms), which gives way to a large rise in [Ca2þ]o during the contraction. At first this result seemed surprising in light of the rabbit results in Fig. 5A. However, when we consider the diVerences in action potential shape and that [Naþ]i is higher in rat ventricular myocytes (Shattock and Bers, 1989), the explanation became clear. The rat (and mouse) ventricle exhibit very short action potential duration compared to
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rabbit (or human ventricle), and this drives rapid Ca2þ extrusion via Naþ/Ca2þ exchange at a time when [Ca2þ]i is very high (Fig. 6B). In the rabbit ventricle, the longer action potential plateau keeps Naþ/Ca2þ exchange in check, delaying extrusion until a later time where [Ca2þ]i is lower. Another implication of Fig. 5B is that there is net Ca2þ eZux during the contraction in rat (vs. net influx in rabbit). This means that there must be net Ca2þ influx between contractions in rat ventricle, and the [Ca2þ]o trace in Fig. 5B is actually going below the bath by the end of the trace to restore the steady state balance before the next beat ( 1.5 s later). Note that during a steady state heartbeat, total Ca2þ influx must equal total Ca2þ eZux (i.e., there is no net gain or loss of Ca2þ at the steady state). Extracellular Ca2þ-MEs are also useful for assessing nonsteady state Ca2þ fluxes on a longer time scale (Bers and MacLeod, 1986; MacLeod and Bers, 1987). Figure 7A shows that when 0.5 Hz stimulation is stopped there is a very slow small rise in [Ca2þ]o over many seconds (net Ca2þ eZux), and upon resumption of stimulation (now at 1 Hz) that there is a net [Ca2þ]o depletion which develops over
B
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Fig. 6 Changes in the reversal potential of the Naþ/Ca2þ exchange (ENa/Ca) during the action potential (Em) and Ca2þ transient in rabbit and rat ventricle. Changes in electrochemical driving force for Naþ/Ca2þ exchange (ENa/Ca Em) are shown in the bottom panels, assuming a 3:1 stoichiometry of Naþ/Ca2þ exchanger and aNai are measured Naþ activity values (Shattock & Bers, 1989). Ca2þ transients driving the contraction are assumed to be the same for both species (resting [Ca2þ]i ¼ 150 nM, peak [Ca2þ]i ¼ 1 mM, 40 ms after the AP initiation). Note that Ca2þ eZux is low during rest in rabbit myocytes because of the low [Ca2þ]i (despite a significant driving force). Based on data in Shattock & Bers, (1989), as modified in Bers (2001), with permission.
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A Control [Ca]o 1 mV
0.2 mM [Ca]o
Tension 200 mN B Ryanodine [Ca]o 1 mV
0.2 mM [Ca]o 30 s
Tension 80 mN
Fig. 7 Measurements of [Ca2þ]o using double-barreled extracellular Ca2þ MEs in rabbit ventricular muscle. Local [Ca2þ]o and tension are shown starting during steady state stimulation at 0.5 Hz with a couple of pauses (0.5 and 1 min) and a period of 1 Hz stimulation. Spikes on [Ca2þ]o trace are stimulus artifacts, and bath [Ca2þ]o was 0.2 mM. The same protocol was used before (A) and after equilibration with 1 mM ryanodine (B; from Bers & MacLeod, (1986), with permission).
several beats. What we showed with other experiments was that this depletion reflects the filling of SR Ca2þ stores (i.e., net transfer of Ca2þ from the extracellular space to the SR). Moreover, the rise in [Ca2þ]o during rest reflects the gradual loss of SR Ca2þ that depends on diastolic leak of Ca2þ from the SR and net Ca2þ extrusion by Na/Ca exchange (thermodynamically favored in resting rabbit ventricular muscle; Fig. 6A). This also allowed new insight at the time as to exactly how ryanodine works in cardiac myocytes. Figure 7B shows that loss of cellular Ca2þ during rest is much faster after ryanodine exposure, but that during 1 Hz pacing the cell (and SR) can take up Ca2þ. This showed that ryanodine makes the SR leaky, but not so much as to abolish SR Ca2þ uptake (i.e., Ca2þ could still be driven into the SR). Once the repeated high [Ca2þ]i signals stop driving SR Ca2þ uptake, the Ca2þ within the SR is lost very quickly. Thus dynamics of cellular Ca2þ flux balance can be readily assessed by double-barreled Ca2þ-MEs, both with relatively high time resolution during steady state conditions and for longer changes that occur in nonsteady state conditions. This makes them a nice complement to fluorescent indicators and voltage clamp studies.
3. Troubleshooting Ca2þ-Selective MEs The ME cannot be filled with the ligand: 1. The ligand may be too thick because of the THF evaporation. Redilute PVC by adding small amounts of THF and stirring the mixture to homogeneity.
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2. The tip diameter is too small. This may entail preparing MEs with diVerent shapes and testing them (ligand filling and calibration) on the same day. Relying on ME resistance may give misleading results because it is also aVected by the geometry of the shank. 3. ME silanization may be insuYcient, because of insuYcient time of silanization (or insuYcient beaker seal during exposure to silane vapors), old silane, glass rehydration (storage problem), and insuYcient dehydration. The ME gives bad calibration curves (subNernstian slopes, low detection limit). 1. Make sure that the calibrating solutions are adequate. Check them with commercial macroelectrodes or the above described ETH 129-based minielectrodes, as described in previously published methods (e.g., Bers, 1982). 2. The ligand may be too old. This is a common occurrence. Ligand lifetime is sensitive to exposure to light and air. 3. The ETH 129 is too diluted. This can occur by not letting THF evaporate enough before ligand filling. In this case, it is better to let all the THF evaporate and then, to re-add small amounts (few tens of microliter) of THF and stir the mixture until a syrup-like solution is obtained. It can also happen if after adding THF to a gelled cocktail, the cocktail is not mixed to homogeneity (by vortexing or sonication).
IV. Discussion We here describe the design and preparation of Ca2þ-selective mini- and microelectrodes, based on the ligand ETH 129 in a PVC matrix. Both mini- and microelectrodes have excellent responses during in vitro calibration with a response time and detection limit superior to that of most commercially available minielectrodes. These electrodes can have multiple applications. We do note, however, that for measuring [Ca2þ]i, the ligand ETH 1001 may be preferable. Other Ca2þ-selective ionophores with a higher selectivity for Ca2þ against other ions, such as K23E1 (Suzuki et al., 1995), have been developed after ETH 129 but their detection limit for calcium is considerably inferior to that of ETH 129-based electrodes (Suzuki et al., 1995). Thus, K23E1 may be useful for clinical analysis of calcium in plasma or other biological fluids, but ETH 129-based electrodes remain superior for calcium measurements in the submicromolar concentration range. ETH 129-based minielectrodes are economical, easy to prepare, and have successfully been used for purposes where the response time of the electrode is appropriate. This includes preparation of calibration solutions, determination of the Kd for EGTA, BAPTA, and oxalate in buVer solutions (Bers, 1982; Harrison and Bers, 1987; Harrison and Bers, 1989; Hove-Madsen and Bers, 1993a), and calibration of indo-1 and furaptra signals cell suspensions (Hove-Madsen and Bers, 1992; HoveMadsen et al., 1998; Shannon and Bers, 1997). The minielectrodes have also been
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used to titrate the passive Ca2þ binding sites in permeabilized myocytes where the cellular Ca2þ uptake and release process are inhibited. The same titration method has also been used to measure total Ca2þ uptake in the SR of permeabilized cardiomyocytes (Hove-Madsen and Bers, 1993a). Ca2þ minielectrodes have also been used together with indo-1 for online measurements of the Ca2þ uptake rate in the SR in permeabilized ventricular myocytes (Hove-Madsen and Bers, 1993b), to examine the eVects of phospholamban phosphorylation and temperature (Hove-Madsen et al., 1998; Mattiazzi et al., 1994) and to measure the number of SR Ca2þ pump sites by titration with the selective pump inhibitor thapsigargin (Hove-Madsen and Bers, 1993b; Hove-Madsen et al., 1998). In these applications, the electrode response time can become a limiting factor and it is important to use a fresh Ca2þ electrode for each experiment, and to minimize inhomogeneities in cell suspensions that the calcium electrodes cannot detect. Although the use of Ca2þ selective electrodes for measurements of dynamic changes in some biological systems is limited by their response time (particularly at submicromolar concentration), the potential benefits of combining Ca2þ selective electrodes with other experimental techniques are underexplored. Indeed, calcium selective electrodes have successfully been used to monitor [Ca2þ] inside patch pipettes (Kang and Hilgemann, 2004; Kang et al., 2003). New Ca2þ sensors based on coating of microcantilevers with ion-selective self-assembled monolayers have also been developed (Ji and Thundat, 2002) and may be useful in the mapping of Ca2þ channels and transporters on the cell surface. Indeed, measurement of the change in extracellular ion concentration with ion-selective MEs has also been shown to provide a noninvasive means for functional mapping of the location and density of potassium channels (Korchev et al., 2000; Messerli et al., 2007) and for the quantification of transmembrane Ca2þ flux (Bers, 1983, 1985, 1987; Bers and MacLeod, 1986; Shattock and Bers, 1989; Smith et al., 1999). Thus, in spite of the overwhelming predominance of fluorescent Ca2þ indicators, Ca2þ-selective electrodes and biosensors still remain a valuable supplement to many imaging and electrophysiological techniques in molecular and cellular physiology. Acknowledgments This work was supported by a grant from the National Institute of Health (HL30077) to DMB and a grant from the Spanish Ministry of Science and Technology (SAF2007-60174) to LHM.
References Allen, P. D., Lopez, J. R., Sanchez, V., Ryan, J. F., and Sreter, F. A. (1992). EU 4093 decreases intracellular [Ca2þ] in skeletal muscle fibers from control and malignant hyperthermia-susceptible swine. Anesthesiology 76, 132–138. Ammann, D. (1986). Ion-Selective Microelectrodes. Principles, Design and Application Springer, Berlin.
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L. Hove-Madsen et al. Ammann, D., Buhrer, T., Schefer, U., Muller, M., and Simon, W. (1987). Intracellular neutral carrierbased Ca2þ microelectrode with subnanomolar detection limit. Pflugers Arch. 409, 223–228. Bers, D. M. (1982). A simple method for the accurate determination of free [Ca] in Ca-EGTA solutions. Am. J. Physiol. 242, C404–C408. Bers, D. M. (1983). Early transient depletion of extracellular Ca during individual cardiac muscle contractions. Am. J. Physiol. 244, H462–H468. Bers, D. M. (1985). Ca influx and sarcoplasmic reticulum Ca release in cardiac muscle activation during postrest recovery. Am. J. Physiol. 248, H366–H381. Bers, D. M. (1987). Mechanisms contributing to the cardiac inotropic eVect of Na pump inhibition and reduction of extracellular Na. J. Gen. Physiol. 90, 479–504. Bers, D. M. (2001) Excitation–Contraction Coupling and Cardiac Contractile Force, 2nd ed., Kluwer Academic Press, Dordrecht, NL. Bers, D. M., and Ellis, D. (1982). Intracellular calcium and sodium activity in sheep heart Purkinje fibres. EVect of changes of external sodium and intracellular pH. Pflugers Arch. 393, 171–178. Bers, D. M., and MacLeod, K. T. (1986). Cumulative depletions of extracellular calcium in rabbit ventricular muscle monitored with calcium-selective microelectrodes. Circ. Res. 58, 769–782. Blatter, L. A., and Blinks, J. R. (1991). Simultaneous measurement of Ca2þ in muscle with Ca electrodes and aequorin. DiVusible cytoplasmic constituent reduces Ca2þ-independent luminescence of aequorin. J. Gen. Physiol. 98, 1141–1160. Blatter, L. A., and Wier, W. G. (1992). Agonist-induced [Ca2þ]i waves and Ca2þ-induced Ca2þ release in mammalian vascular smooth muscle cells. Am. J. Physiol. 263, H576–H586. Deyhimi, F., and Coles, J. A. (1982). Rapid silylation of a glass surface: Choice of reagent and eVect of experimental parameters on hydrophobicity. Helv. Chim. Acta 65, 1752–1759. Fedirko, N., Svichar, N., and Chesler, M. (2006). Fabrication and use of high-speed, concentric Hþ- and Ca2þ-selective microelectrodes suitable for in vitro extracellular recording. J. Neurophysiol. 96, 919–924. Gorman, A. L., Levy, S., Nasi, E., and Tillotson, D. (1984). Intracellular calcium measured with calcium-sensitive micro-electrodes and Arsenazo III in voltage-clamped Aplysia neurones. J. Physiol. 353, 127–142. Grynkiewicz, G., Poenie, M., and Tsien, R. Y. (1985). A new generation of Ca2þ indicators with greatly improved fluorescence properties. J. Biol. Chem. 260, 3440–3450. Harkins, A. B., Kurebayashi, N., and Baylor, S. M. (1993). Resting myoplasmic free calcium in frog skeletal muscle fibers estimated with fluo-3. Biophys. J. 65, 865–881. Harrison, S. M., and Bers, D. M. (1987). The eVect of temperature and ionic strength on the apparent Ca-aYnity of EGTA and the analogous Ca-chelators BAPTA and dibromo-BAPTA. Biochim. Biophys. Acta 925, 133–143. Harrison, S. M., and Bers, D. M. (1989). Correction of proton and Ca association constants of EGTA for temperature and ionic strength. Am. J. Physiol. 256, C1250–C1256. Hove-Madsen, L., and Bers, D. M. (1992). Indo-1 binding to protein in permeabilized ventricular myocytes alters its spectral and Ca binding properties. Biophys. J. 63, 89–97. Hove-Madsen, L., and Bers, D. M. (1993). Passive Ca buVering and SR Ca uptake in permeabilized rabbit ventricular myocytes. Am. J. Physiol. 264, C677–C686. Hove-Madsen, L., and Bers, D. M. (1993). Sarcoplasmic reticulum Ca2þ uptake and thapsigargin sensitivity in permeabilized rabbit and rat ventricular myocytes. Circ. Res. 73, 820–828. Hove-Madsen, L., Llach, A., and Tort, L. (1998). Quantification of Ca2þ uptake in the sarcoplasmic reticulum of trout ventricular myocytes. Am. J. Physiol. 275, R2070–R2080. Ji, H. F., and Thundat, T. (2002). In situ detection of calcium ions with chemically modified microcantilevers. Biosens. Bioelectron. 17, 337–343. Kang, T. M., and Hilgemann, D. W. (2004). Multiple transport modes of the cardiac Naþ/Ca2þ exchanger. Nature 427, 544–548. Kang, T. M., Markin, V. S., and Hilgemann, D. W. (2003). Ion fluxes in giant excised cardiac membrane patches detected and quantified with ion-selective microelectrodes. J. Gen. Physiol. 121, 325–347.
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Konishi, M., Olson, A., Hollingworth, S., and Baylor, S. M. (1988). Myoplasmic binding of fura2 investigated by steady-state fluorescence and absorbance measurements. Biophys. J. 54, 1089–1104. Korchev, Y. E., Negulyaev, Y. A., Edwards, C. R., Vodyanoy, I., and Lab, M. J. (2000). Functional localization of single active ion channels on the surface of a living cell. Nat. Cell. Biol. 2, 616–619. Levy, S., and Fein, A. (1985). Relationship between light sensitivity and intracellular free Ca concentration in Limulus ventral photoreceptors. A quantitative study using Ca-selective microelectrodes. J. Gen. Physiol. 85, 805–841. Lipp, P., Luscher, C., and Niggli, E. (1996). Photolysis of caged compounds characterized by ratiometric confocal microscopy: A new approach to homogeneously control and measure the calcium concentration in cardiac myocytes. Cell Calcium 19, 255–266. Lopez, J. R., Contreras, J., Linares, N., and Allen, P. D. (2000). Hypersensitivity of malignant hyperthermia-susceptible swine skeletal muscle to caVeine is mediated by high resting myoplasmic [Ca2þ]. Anesthesiology 92, 1799–1806. MacLeod, K. T., and Bers, D. M. (1987). EVects of rest duration and ryanodine on changes of extracellular [Ca] in cardiac muscle from rabbits. Am. J. Physiol. 253, C398–C407. Marban, E., Rink, T. J., Tsien, R. W., and Tsien, R. Y. (1980). Free calcium in heart muscle at rest and during contraction measured with Ca2þ-sensitive microelectrodes. Nature 286, 845–850. Mattiazzi, A., Hove-Madsen, L., and Bers, D. M. (1994). Protein kinase inhibitors reduce SR Ca transport in permeabilized cardiac myocytes. Am. J. Physiol. 267, H812–H820. Messerli, M. A., Corson, E. D., and Smith, P. J. (2007). Measuring extracellular ion gradients from single channels with ion-selective microelectrodes. Biophys. J. 92, L52–L54. Orchard, C. H., Boyett, M. R., Fry, C. H., and Hunter, M. (1991). The use of electrodes to study cellular Ca2þ metabolism. In ‘‘Cellular Calcium. A Practical Approach,’’ (J. G. McCormack, and P. H. Cobbold, eds.), Oxford University Press, New York. Picht, E., DeSantiago, J., Blatter, L. A., and Bers, D. M. (2006). Cardiac alternans do not rely on diastolic sarcoplasmic reticulum calcium content fluctuations. Circ. Res. 99, 740–748. Purves, R. D. (1981). Microelectrode Methods for Intracellular Recording and Ionophoresis. Academic Press, New York. Schefer, U., Ammann, D., Pretsch, E., Oesch, U., and Simon, W. (1986). Neutral carrier based Ca2þselective electrode with detection limit in the subnanomolar range. Anal. Chem. 58, 2282–2285. Shannon, T. R., and Bers, D. M. (1997). Assessment of intra-SR free [Ca] and buVering in rat heart. Biophys. J. 73, 1524–1531. Shattock, M. J., and Bers, D. M. (1989). Rat vs. rabbit ventricle: Ca flux and intracellular Na assessed by ion-selective microelectrodes. Am. J. Physiol. 256, C813–C822. Smith, P. J. S., Hammar, K., Porterfield, D. M., Sanger, R. H., and Trimarchi, J. R. (1999). Selfreferencing, non-invasive, ion selective electrode for single cell detection of trans-plasma membrane calcium flux. Microsc. Res. Tech. 46, 389–417. Suzuki, K., Watanabe, K., Matsumoto, Y., Kobayashi, M., Sato, S., Siswanta, D., and Hisamoto, H. (1995). Design and synthesis of calcium and magnesium ionophores based on double-armed diazacrown ether compounds and their application to an ion sensing component for an ion-selective electrode. Anal. Chem. 67, 324–334. Thomas, R. C. (1982). Ion-Selective Microelectrodes: How to Make and Use Them. Academic Press, New York. Tsien, R. Y., and Rink, T. J. (1981). Ca2þ-selective electrodes: A novel PVC-gelled neutral carrier mixture compared with other currently available sensors. J. Neurosci. Methods 4, 73–86. Ujec, E., Keller, O., Machek, J., and Pavlik, V. (1979). Low impedance coaxial Kþ selective microelectrodes. Pflugers Arch. 382, 189–192. Vaughan-Jones, R. D., and Kaila, K. (1986). The sensitivity of liquid sensor, ion-selective microelectrodes to changes in temperature and solution level. Pflugers Arch. 406, 641–644. Vaughan-Jones, R. D., and Wu, M. L. (1990). pH dependence of intrinsic Hþ buVering power in the sheep cardiac Purkinje fibre. J. Physiol. 425, 429–448.
CHAPTER 4
Construction, Theory, and Practical Considerations for using Self-referencing of Ca2þ-Selective Microelectrodes for Monitoring Extracellular Ca2þ Gradients Mark A. Messerli and Peter J. S. Smith BioCurrents Research Center Cellular Dynamics Program Marine Biological Laboratory Woods Hole, Massachusetts, USA
Abstract I. Introduction II. CaSM Construction A. Micropipette Fabrication B. Silanization C. Microelectrode Construction III. Properties of CaSMs A. Response to Ion Activity B. Selectivity C. Spatial Resolution D. Response Time IV. Self-referencing of CaSMs A. DiVerential Concentration Measurement B. DiVerential Concentration Determination C. Calculation of Flux D. Correction for Ca2þ BuVering E. Measurement of Voltage Gradients F. Positional Artifacts V. Ca2þ Flux Measurements References
METHODS IN CELL BIOLOGY, VOL. 99 Copyright 2010, Elsevier Inc. All rights reserved.
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0091-679X/10 $35.00 DOI: 10.1016/S0091-679X(10)99004-3
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Mark A. Messerli and Peter J. S. Smith
Abstract Ca2þ signaling in the extra- and intracellular domains is linked to Ca2þ transport across the plasma membrane. Noninvasive monitoring of these resulting extracellular Ca2þ gradients with self-referencing of Ca2þ-selective microelectrodes is used for studying Ca2þ signaling across Kingdoms. The quantitated Ca2þ flux enables comparison with changes to intracellular [Ca2þ] measured with other methods and determination of Ca2þ transport stoichiometry. Here, we review the construction of Ca2þ-selective microelectrodes, their physical characteristics, and their use in self-referencing mode to calculate Ca2þ flux. We also discuss potential complications when using them to measure Ca2þ gradients near the boundary layers of single cells and tissues.
I. Introduction Regulation of resting [Ca2þ]i and the control of spatial and temporal dynamics during Ca2þ signaling require coordinated transport between membraneseparated compartments, giving rise to Ca2þ fluxes across organelles and the plasma membrane. Movement of Ca2þ across the plasma membrane via transporters, exchangers, or channels gives rise to minute gradients of [Ca2þ] in the extracellular boundary layer that reflect changes in [Ca2þ]i. The near real-time extraction of these gradients requires a detection method that is not disturbing to the local chemical environment, functions over a wide dynamic range, and possesses high sensitivity, selectivity, and spatial resolution. For these reasons extracellular Ca2þ gradients have been monitored with self-referencing of Ca2þselective microelectrodes (CaSMs), enabling noninvasive characterization of Ca2þ transport and signaling events. Unlike most fluorescent or luminescent indicators, CaSMs were originally developed for measuring both intracellular and extracellular [Ca2þ] (listed in Lanter et al., 1982). Measurement of minute Ca2þ gradients on the outside of cells was limited by electrical drift in the system. For this reason, a modulation technique was introduced (Ku¨htreiber and JaVe, 1990) that enabled reduction of drift and provided a simple means for calculating Ca2þ flux. The method was later coined ‘‘self-referencing’’ and has been extended to other ion-selective microelectrodes and amperometric microelectrodes enabling characterization of fluxes of many diVerent analytes (Messerli et al., 2006; Smith et al., 2007). Measurement of Ca2þ fluxes with selfreferencing has enabled direct comparison of Ca2þ fluxes measured with other techniques including radioactive tracers, fluorescent and luminescent ion indicators, and voltage clamp. We will first discuss the construction and general properties of CaSMs before discussing their use with the self-referencing approach.
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II. CaSM Construction A. Micropipette Fabrication Ion-selective microelectrodes are based on an ion-selective solvent or liquid membrane, immobilized in the tip of a glass micropipette with a backfilling electrolyte. The glass micropipette housings are pulled from 1.5 mm outer diameter borosilicate (TW150-4 World Precisions Instruments, Sarasota, FL), aluminosilicate (A150-100-10 Sutter Instruments, Novato, CA), or quartz glass (Q150-110-10, Sutter Instruments). Inner filaments, commonly used to load electrolyte solutions to the tips of micropipettes, are avoided. Although the glass body is fragile, it provides distinct advantages over other materials including low cost, excellent resistive properties necessary for use with the high-resistance liquid membranes, and easy fabrication of small tips. Micropipettes are pulled, silanized, and stored in bulk, 50 per wire rack. The glass is pulled down to a final edge slope of 0.15–0.17 and an inner tip diameter of 2–3 mm. Borosilicate and aluminosilicate micropipettes are pulled on a horizontal heated filament puller (P-97, Sutter Instruments) while quartz pipettes are pulled on a horizontal laser puller (P-2000, Sutter Instruments). Latex gloves are worn during handling of the glass before silanization.
B. Silanization The hydrophilic glass surfaces are coated with a hydrophobic silane to enable adhesion and high electrical resistance between the glass and the hydrophobic liquid membrane. While many forms of silanization exist, we prefer vapor deposition of N,N-dimethyltrimethylsilylamine (cat# 41716 Sigma-Aldrich, St. Louis, MO) as it enables rapid and uniform coating of numerous micropipettes, simultaneously. A wire rack of micropipettes is placed in a small solid wall metal box (8 cm 8 cm 10 cm) with a swinging door within the oven so that the silane vapor can be trapped in a small region around the pipettes. Prior to coating, the glass is dried for 20 min at 240 C under vacuum (28 in Hg). This shortens drying time and decreases loss of hydroxyl groups (Deyhimi and Coles, 1982; Munoz et al., 1983). Higher temperatures may dry glass more quickly as well; however, this silane has ignited two out of four times at 250 C. After drying, atmospheric pressure is recovered by purging the oven with Argon. A small volume of silane (20 mL) is dropped into a tiny glass beaker in the metal enclosure and the door to the enclosure is closed before the oven door is closed. The glass is exposed to the silane vapor for 20 min before removing and placing the micropipettes in a sealed bell jar with desiccant in the bottom. Functional CaSMs have been produced from micropipettes that have been stored in this manner for up to a month. This method has reduced variation in the quality of silanization.
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C. Microelectrode Construction Standard, electrolyte-based CaSMs consist of a short column of liquid membrane ( 30 mm) with a longer column of Ca2þ containing electrolyte (5 mm) used to make electrical contact with the voltage recording headstage via a Ag/AgCl wire. Commercially available vented pipette holders (WPI, Sarasota, FL, Warner Instruments, Hamden, CT) are used to immobilize the CaSMs while loading and recording from the high input impedance electrometers 1015 Ω (BioCurrents Research Center, Woods Hole, MA; Molecular Devices, Sunnyvale, CA; Warner Instruments, Hamden, CT). CaSMs are constructed by first backfilling a few millimeters of the electrolyte into a silanized micropipette with a long blunt needle and syringe, before tip loading the liquid membrane. The backfilling electrolyte has varied from 100 nM Ca2þ buVered with 5 mM EGTA, 10 mM HEPES with 90 mM KCl (Tsien and Rink, 1981) to simply 100 mM CaCl2 (Ku¨htreiber and JaVe, 1990). However, based on further discussion below it will be shown that the backfilling solution should be based on the bath [Ca2þ] with additional electrolyte, 100 mM KCl, to make electrical contact with the Ag/AgCl wire. Ca2þ-selective liquid membranes can be mixed in the lab or purchased premixed (cat# 21048 (ETH1001), cat# 21196 (ETH129); Sigma-Aldrich, St. Louis, MO). Tip loading of the liquid membrane is performed under microscopic control, displayed in Fig. 1A. The electrolyte filled micropipette on the right is positioned near a loading pipette on the left, a tip broken micropipette that has been dip-loaded with liquid membrane. Both the loading pipette and the CaSM are connected to airfilled syringes with plastic tubing so that pressure can be applied. The threaded plunger (TP) syringe in Fig. 1A allows a small controlled pressure to create a small bulge of liquid membrane away from the loading pipette which aids loading of the CaSM. A plastic syringe (PS) with a three-way valve for the CaSM enables applying and venting pressure before loading and before removing the CaSM from the electrode holder. After positioning both the loading pipette and the CaSM within the field of view under the microscope objective, Fig. 1B, pressure is applied to the back of the CaSM to push the electrolyte to the tip. Pressure is vented and the tip of the CaSM is immediately positioned within the liquid membrane bulge held in the loading pipette. Surface tension will immediately draw the liquid membrane into the silanized micropipette. A combination of pressure and suction is used to achieve a liquid membrane column of the desired length ( 30 mm). After the desired length is achieved, the CaSM tip is removed from the liquid membrane, the back of the CaSM is vented to atmospheric pressure, and the CaSM is removed from its holder.
III. Properties of CaSMs A. Response to Ion Activity The potential across the Ca2þ-selective liquid membrane in the tip of the CaSM is comprised of two phase boundary potentials, between the interfaces of the liquid membrane with (1) the backfilling solution and (2) the extracellular medium, and
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A
Loading pipette
CaSM
Loading pipette
CaSM
TP PS
Fig. 1 Ca2þ-selective microelectrode tip filling station. (A) Micropositioners on each side of an upright microscope are used to position the tips of a loading pipette and a CaSM in the field of view. The stage has been removed. The threaded plunger (TP) syringe and the plastic syringe (PS) are connected to the loading pipette and CaSM via plastic tubing enabling application of pressure and suction to control the length of liquid membrane loaded into the CaSM from the loading pipette. (B) Higher magnification of A showing the close positioning of the glass loading pipette and glass CaSM. The system is mounted on a large metal plate to reduce vibration during loading.
also the diVusion potential between the two ends of the column of liquid membrane (Bakker et al., 1997). The diVusion potential is considered negligible as bulk movement of Ca2þ across the liquid membrane does not occur during common usage with high-impedance electronics and no current flow. The inner phase boundary potential is considered constant due to the rigorous clamping of Ca2þ with buVers or with high concentrations of Ca2þ in the backfilling electrolyte. The external phase boundary potential, for an ideal ion-selective microelectrode, is related to the extracellular ion activity by the Nernst equation, E ¼ EO þ S log ai
ð1Þ
where ‘‘Eo’’ is the sum of constant potential contributions, ‘S’is the Nernstian slope ¼ (2.3RT) / (ziF) (R, T, and F hold their standardized meanings) and ‘‘ai’’ is the activity of the primary ion. Constant potential contributions are comprised of the boundary potentials and liquid junction potentials that exist across the circuit comprising the reference and measuring electrodes. The valence ‘‘zi’’ of Ca2þ produces a slope only half as steep ( 29 mV/order magnitude change in Ca2þ) compared to monovalent ions. The high selectivity of the two Ca2þ liquid membranes discussed here along with the generally standard physiological media that are used enables us to perform flux
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calculations according to the Nernst equation listed above. However, in complex media with significantly interfering ions, the slope of response can be reduced. The decrease in slope can be predicted using the Nicolsky–Eisenman equation for ions of similar valence and the extended Nicolsky–Eisenman equations for ions of diVerent valence (Bakker et al., 1994). In some cases it may be more practical to perform an empirical determination of the slope of the line describing the relationship between measured voltage and the change in ionic activity. This determination is performed by making up the working medium with slightly higher and lower concentrations of Ca2þ and determining the slope of response. A sub-Nernstian response may reflect the presence of an interfering ion or of a substance that is fouling the microelectrode. According to the Nernst equation, the voltage output is dependent on ionic activity. However, as ion activity is directly proportional to ion concentration, via the activity coeYcient, and the changes that occur to the activity coeYcient due to changes in ionic strength are negligible during self-referencing in physiological saline, we will use concentration in place of activity for further discussion.
B. Selectivity A primary motivation for early development of CaSMs was to monitor intracellular [Ca2þ] (Lanter et al., 1982; Tsien and Rink, 1981). This required a liquid membrane with high selectivity for monitoring the low resting [Ca2þ]i ( 100 nM) in the presence of higher concentrations of potentially interfering ions including Kþ ( 120 mM), Naþ ( 10 mM) and Mg2þ ( 1 mM). Accordingly, two diVerent Ca2þ ionophores with very high selectivity were reported (Ammann et al., 1975, 1987; Lanter et al., 1982). Some of their selectivity coeYcients for Ca2þ over other common cations are listed in Table I. Selectivity for Ca2þ over these cations is relatively good compared to liquid membranes for other ions. However, not all inorganic or organic ions have been tested and may therefore act as interferents. Not only do interfering ions reduce the electrode’s voltage response to the primary ion but they also slow the response time of the electrode (Bakker et al., 1997). This point is particularly important when using the electrodes in self-referencing mode where a temporal component is part of the modulation approach. In biological applications, it is critical for the investigator to empirically test the voltage response of a CaSM in the medium in which the experiments are to be performed. Simple solutions of the primary ion are not suYcient. Additionally, the CaSM should be tested for interference or fouling due to the addition of transport blockers or cellular poisons.
C. Spatial Resolution Small, micron-sized sensors give rise to high spatial resolution. The spatial resolution is defined first by the external surface area of the Ca2þ-selective liquid membrane, but also by the sampling time and the distance between the source of the Ca2þ transport and the CaSM. This holds true for the high-impedance
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Table I Selectivity coeYcients of two diVerent Ca2þ-selective liquid membranes Selectivity coeYcients (log CaMPot) Interfering ion (M) Kþ Naþ Mg2þ NH4þ Hþ
ETH1001
ETH129a
5.4b 5.5b 4.9b 5.0c 4.4c
7.2 5.8 6.7 3.6 2.5
a
Ammann et al. (1987). Lanter et al. (1982). c Ammann et al. (1975). b
headstages 1015 Ω that are typically used, which help to decrease the bulk movement of Ca2þ between the medium and liquid membrane. Spatial resolution is decreased due to diVusion of Ca2þ in the bulk medium from nearby transport events. DiVusion of Ca2þ from 10 to 20 mm away will reach the CaSM in only 20 and 80 ms, indicating that the sampled volume is much larger than the immediate dimensions of the CaSM tip. As these events are diVusing from regions further away, the local concentration change that they produce near the tip of the CaSM will be much smaller (proportional to 1/r2) than the signals from events immediately in front of the CaSM. The decay in signal with distance is evident from measurements of extracellular Kþ gradients due to eZux through single Kþ channels (Messerli et al., 2009). The sampling domain of the CaSM is therefore slightly larger than the surface area of the liquid membrane and decays rapidly with increasing distance from the surface.
D. Response Time Self-referencing of CaSMs requires the use of CaSMs with relatively short response times so that the CaSM can reach equilibrium in a short period of time at its new position. The response time of CaSMs is governed by the ability to provide charge to the sensing node. In an ideal measuring system, diVusion through the unstirred layer at the surface of the electrode defines the response time of the sensors when the liquid membrane is equilibrated with the salt of an ion to which the electrode responds (Bakker et al., 1997). For ion-selective microelectrodes, this process may occur so quickly that the electronics of the system slow the measured response (Ammann, 1986). Low input impedance of the amplifier and parasitic capacitances in the circuit will draw more charge than an ideal system therefore slowing the response time of the system. Amplifier input impedances of
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Table II CaSMs based on ionophore ETH1001 possess short response times in physiological saline over a range of [Ca2þ] Response times (t95% ms) 0.1–1 mM 48 7
1–10 mM 53 10
10–1 mM 58 9
1–0.1 mM 81 10
Physiological saline consists of (in micromolar) 120 NaCl, 5 KCl, 2 MgCl2, 10 HEPES with the CaCl2 concentration listed above. CaSMs remained stationary during the experiment, while three adjacent streams of media (1 mL/min) were rapidly positioned (<8 ms) in front of the measuring electrode. These measurements describe the response time of the entire measuring system for 4 CaSMs.
106 GO are typically used to accommodate ion-selective microelectrodes that have high resistances, 1–20 GO (Ammann, 1986). Even with the best electronics, the time constant (RC, resistance times capacitance) of the CaSM itself imposes a low pass filter. Resistance is primarily dependent on the tip diameter and length of the column of liquid membrane and capacitance is primarily dependent on the thickness and dielectric constant of the wall of the glass micropipette. To reduce the resistance of the CaSMs, they are fabricated with relatively large tips of 2–3 mm inner diameter and with short columns of liquid membrane 30 mm. The short columns are achieved by tip loading the liquid membrane as discussed above. Capacitance can be lowered by using thicker walled borosilicate glass (1.5 mm O. D. 0.84 mm I.D. cat# 1B150-6, WPI Sarasota, FL). The construction design listed above has produced CaSMs with response times shorter than 100 ms, Table II. In practice, a slight deviation from the expected length does not change the response time very much, at least when considering the use of these electrodes in the self-referencing application.
IV. Self-referencing of CaSMs A. DiVerential Concentration Measurement Self-referencing of CaSMs was developed to measure extracellular Ca2þ gradients/currents that may have existed near previously characterized extracellular voltage gradients (Ku¨htreiber and JaVe, 1990). For example, relatively steady eZux of Ca2þ across the plasma membrane gives rise to a gradient of Ca2þ with a higher concentration near the cell. Self-referencing of CaSMs is implemented by measuring the [Ca2þ] at two points in that Ca2þ gradient. The electrical variation of a single CaSM due to thermal noise, 100–200 mV, of the high-impedance sensors and chemical drift is too large to enable measurement of such small diVerences in extracellular Ca2þ. As a result, a frequency sensitive method of
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detection was explored based on response times of about 1 s reported for CaSMs commonly used at that time (Ammann, 1986). The general measuring protocol includes intermittent collection of ion concentration by a single CaSM at a position near the biological preparation and then at a position some distance away orthogonal to the source. A CaSM is shown in Fig. 2A and B at the two positions, next to a mouse pancreatic islet. The excursion distance in this case is 20 mm but can vary between 5 and 50 mm depending on the size of the cell or cellular preparation. At each pole of excursion, the CaSM is allowed to reach equilibrium ( 0.25 s) before recording the average local ion concentration for about 1 s. Considering that the CaSMs possess response times of less than 0.1 s, 0.25 s is plenty of time to reach equilibrium. The CaSM is then immediately positioned to the opposite pole, and allowed to reach equilibrium before recording the local ion concentration. The movement of the CaSM between the two positions is controlled by stepper motors set to move the sensor at a rate of 40 mm/s such that it takes 0.25 s to reach its new
A
B
Near pole (E1) Far pole (E2) 50 mm C
ΔE = E1 − E2
Near pole ΔE
E2
d
E1
d
E2
d
E1
d
Far pole
Fig. 2 Ca2þ flux measurements performed with self-referencing of a Ca2þ-selective microelectrode near a mouse pancreatic islet. (A) In the near pole the CaSM collects the average [Ca2þ]-dependent potential for 1 s, E1. (B) After movement to the far pole and equilibration, the average [Ca2þ]-dependent potential is collected again for 1 s, E2. (C) Data collection scheme portraying Ca2þ eZux. The automated determination of the diVerential [Ca2þ]-dependent potential, DE, is used to determine Ca2þ flux. This measuring scheme continues, as defined by the user. The [Ca2þ]-dependent potential is discarded, (d), during periods of movement (0.25 s) and during equilibration in the new position (0.25 s).
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position. A diVerential concentration recording between the two poles of excursion is collected, about every 3 s. The measurement scheme continues until stopped by the user. The diVerential recording possesses peak to peak noise of 10 mV while longer periods of signal collection and averaging can enable extraction of concentration diVerences that give rise to 1 mV diVerences or a 0.008% diVerence from the background [Ca2þ]. Figure 2C illustrates this collection scheme during Ca2þ eZux where E1 and E2 are the recorded ion concentration–dependent potentials at the two poles. The recording collected during movement and equilibration at the new pole is discarded, labeled ‘‘d’’ in Fig. 2C. A diVerential Ca2þ measurement is collected over a period of about 3 s, which is faster than the low frequency drift, thus reducing its influence on the measurement. Signal averaging at each pole over a period of 1 s reduces the influence of the high frequency noise. Measurement of the diVerential ion concentration–dependent voltage, DE, between the two positions over time enables further enhancement of the signal-to-noise ratio. This modulation approach was termed self-referencing, referring to the fact that the measurements, collected by a single CaSM, are compared to each other in order to determine the concentration diVerence between the two points. The signal collected by a single CaSM at one point in space and time is referenced to the signal collected by that same CaSM at a diVerent point in space and time in order to reduce electrical drift of the measuring system. The CaSM has its own bath reference electrode. While this diVerential measurement could be achieved with two similar, CaSMs, positioned at known distances from the source, the sensitivity would suVer from the signal drift and noise inherent to two separate measuring systems. Measured diVerential voltages of 10s mV are extracted from relatively large oVset potentials 100s mV by using a combination of amplification methods. Low gain must be used with the large oVset potentials in order to keep the signal within the dynamic range of the amplifier. As a result, low resolution digital systems will not be able to register small changes in the diVerential voltage. A 12-bit system with a dynamic range of 10 V provides only 4.9 mV/bit resolution while a 16-bit system provides only 0.3 mV/bit. Additional amplification prior to digitization is necessary to resolve signals at or below 1 mV. Two separate methods for amplification are used; (1) a nearly equal and opposite electrical oVset is supplied before amplification (sample hold mode) and (2) a running average of the low gain measurement is subtracted from the real-time input before amplification (RC subtract mode). Sample hold mode applies a known voltage that is selected either manually or automatically from the signal after a set duration of time to null the oVset potential before applying 103 times gain. The primary disadvantage for this mode is that drift can take the system back out of the dynamic range of the amplifier so that a new potential must be applied regularly. The advantage is that it does not need an additional correction factor to compensate for the signal lost due to the filtering that occurs in RC subtract mode. In RC subtract mode, a high-pass filter is used to collect a running average potential that is subtracted from the potentials collected in the near and far pole. The signals are then amplified
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103 times before digitizing. Typically, this mode employs a high-pass filter with a time constant of 10 s. RC subtract allows amplification for systems with large drift but involves a correction factor to oVset the high-pass filter. The correction factor will be dependent on the time constant of the high-pass filter and the period of data acquisition. For standard conditions, a period of 3.3 s (0.3 Hz translation frequency), 40 mm/s translation speed, 10 s time constant of the high-pass filter along with data selection of the last 70% of the half cycle, we calculate that the signal is 7% smaller than a square wave with similar rise time. Automated, repetitive positioning of the CaSMs is controlled by three stepper motors arranged in an X, Y, Z configuration with the Z plane parallel to the plane of the stage of the microscope. Smooth linear motion is obtained by coupling each of the stepper motors to a lead screw controlling the position of three small translational plates connected together to form a three-dimensional positioner (BioCurrents Research Center, Woods Hole, MA). Low voltage control of the stepper motors prevents electrical feedback to the high-impedance headstage of the CaSM. Positioning can be achieved over a working distance of 3–4 cm with submicron resolution and repeatability (Danuser, 1999). A computer interface enables repetitive motion and positional control with the Faraday cage closed.
B. DiVerential Concentration Determination The relationship between the measured diVerential voltage and the diVerential ion concentration between the two poles of excursion for an ideal CaSM can be determined using the Nernst equation. E1 E2 ¼ ðEO þ S log Ci Þ1 ðEO þ S log Ci Þ2 DE ¼ ðS log Ci Þ1 ðS log Ci Þ2 DE ¼ log CiSð11 Þ log!CiSð22 Þ CiSð11 Þ DE ¼ log CiSð22 Þ
ð2Þ
‘‘E1’’, ‘‘Ci(1)’’, and ‘‘S1’’ are the measured voltage, [Ca2þ] and slope of the voltage– log(Ci) graph for the near pole of excursion. The subscript 2 labels the same parameters for the far pole of excursion. The slow changing constant potential contributions ‘‘Eo’’ are reduced if not eliminated by calculating the diVerence between potentials over short periods of time. Equation (2) enables a clear picture of the relationship of the sensitivity of detection to the background ion concentration during measurements. For a given [Ca2þ] change due to cellular flux, the concentration in the position next to the cell, Ci(1), is the sum of the background ion concentration and the concentration change generated by the source while Ci(2), in most cases, is close to the background ion concentration. It is easier to generate a larger DE when the background concentration of the measured ion is lower as the ratio of Ci(1)/Ci(2)
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will be much larger/smaller for the same Ca2þ eZux/influx on lowered background [Ca2þ]. This has led to the lowering of the background [Ca2þ] in order to generate DE with a greater signal-to-noise ratio, see Table IV. Care must be taken to ensure that changing the background concentration does not interfere with normal cellular activity. Rearrangement of Eq. (2) relates the [Ca2þ] in the near pole of excursion to the [Ca2þ] at the far pole of excursion. S2 S
DE
1 Cið1Þ ¼ Cið2Þ 10 S1
ð3Þ
For an ideal electrode, the voltage output is close to the Nernstian slope over a wide range of [Ca2þ] so S1 ¼ S2 ¼ S ¼ 2:3RT zi F . Therefore, Eq. (3) simplifies to DE
Cið1Þ ¼ Cið2Þ 10 S
ð4Þ
For minute fluxes that are typically measured with self-referencing, the average concentration of Ca2þ at the far pole, position 2, is not too diVerent from the average concentration of Ca2þ in the bulk solution. Therefore the diVerence in [Ca2þ] between the two points of excursion can be described as follows: DE
DC ¼ Cið1Þ Cið2Þ ¼ Cbath 10 S Cbath
ð5Þ
A primary assumption here is that the excursion distance is small compared to the extent that the gradient extends out into the bulk solution so that the concentration diVerence between the two excursion points is linear. For minute gradients measured from small cells ( 10 mm diameter), an excursion of 10 mm will most likely sample over a distance in which the concentration diVerence is not linear and therefore will lead the investigator to underestimate the true flux. Incorrect estimation of the true flux could also occur during a two-point measurement in a more intense, extended gradient, where the concentration of the ion in the far pole is substantially diVerent from the background concentration of the ion. In both of these cases, a three-point measurement should be performed in order to (1) more carefully map the concentration gradient with a third point to ensure a linear relationship or determine a more accurate nonlinear relationship and (2) to determine the concentrations in the gradient relative to the background concentration of the ion in the bath. The selectivity of Ca2þ liquid membranes is relatively good compared to liquid membranes for other ions. Therefore, measurement of Ca2þ gradients in the presence of a constant concentration of an interfering ion or in the presence of a gradient of an interfering ion is not a major concern. However, specific circumstances may require the use of higher concentration of an interfering ion and the two cases need to be addressed. Details necessary to account for these situations have been addressed previously (Messerli et al., 2006; Smith et al., 2007).
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C. Calculation of Flux The diVerential concentration measurement is converted to flux to provide a direct representation of the number of ions passing through a unit area per unit time. Calculation of flux enables comparison of Ca2þ transport between diVerent systems as it takes into account the diVusion coeYcient of Ca2þ, the distance over which the diVerential concentration measurement was acquired, the surface geometry of the source, and the distance of measurement from the source. It also provides a value for comparison of Ca2þ flux measured with self-referencing of CaSMs to other methods for monitoring Ca2þ including intracellular fluorescent and luminescent ion indicators and radioactive tracer flux studies. For planar sources where the measuring electrode is relatively close to a large source, such as a tissue, sheet of cells or large diameter cell, and the diVerential concentration is measured over a small distance ‘‘Dx’’ within the gradient next to the source, flux (J) is J ¼ D
DC Dx
ð6Þ
where ’’D’’ is the diVusion coeYcient of Ca2þ. By this model, at equilibrium the flux measured at some distance from the source is the same as the flux at the surface of the source. According to this equation, eZux, a higher concentration of Ca2þ near the source, is identified by a negative flux. In order to determine flux at the cell surface for known surface geometries, it is useful to calculate analyte flow, that is, the quantity of substance (Q) moving per unit time (Henriksen et al., 1992). Flow is the same for all concentric surfaces surrounding the source surface. Flux at the source surface is the flow divided by the surface area of the source. Therefore, radially emanating flow from a cylindrical surface is Flow ¼
Q 2pD ¼ ðDC Þ t lnðb=aÞ
ð7Þ
where ‘‘D’’ is the diVusion coeYcient of the analyte and ‘‘a’’ and ‘‘b’’ are the radial distances between the center of the cylinder and the electrode tip at the near and far poles, respectively. These equations have been adapted from Crank (1967). Analyte flux at the surface of the cylinder is then determined by dividing by its surface area 2prl. A caveat of this approach is the assumption that the flow is equal at all points around the cylinder and along the shaft of the cylinder. An alternative is to calculate flux per unit length by dividing by 2pr (Henriksen et al., 1992). The flow from a spherical source is Flow ¼
Q ab ¼ 4pD ðDC Þ t ba
ð8Þ
Flux at the cell surface can then be determined by dividing by the spherical surface area 4pr2.
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D. Correction for Ca2þ BuVering The presence of Ca2þ buVers or binding agents with the appropriate aYnity can lead to collapse of Ca2þ gradients by shuttle buVering (Speksnijder et al., 1989). Ca2þ can diVuse from the surface of the cell in either its free state or bound to the buVer. CaSMs only measure the free concentration of Ca2þ. The actual Ca2þ flux at a source is the sum of the measured free Ca2þ flux and the unmeasured Ca2þ flux diVusing as Ca2þ bound to buVer. JCa
total
¼ JCa
measured
þ JCa
Buffer
ð9Þ
2þ
Knowing the conditions under which the Ca flux was measured including the [Ca2þ] of medium, dissociation constant, Kd, of the buVers and concentration of the buVers present, a simple relationship can be derived to determine the ratio of Ca2þ diVusing bound to buVer compared to the freely diVusing Ca2þ. Shuttle buVering of Hþ is a bigger concern than for Ca2þ due to the larger number of Hþ buVers that are used in physiological media. The equations that exist to correct for shuttle buVering of Hþ (Messerli et al., 2006; Smith et al., 2007) can be adapted for use with Ca2þ flux correction and may be necessary under specific circumstances. xi ¼
DB Kd ½B 2 DCa2þ Kd þ Ca2þ
ð10Þ
The correction factor, ‘‘xi’’, is the ratio of the Ca2þ bound buVer flux to the free Ca2þ flux. Therefore JCa
total
¼ JCa
measured ð1
þ xi þ þ x n Þ
ð11Þ
where a number of diVerent Ca buVers (xi þ þ xn) may be collapsing the Ca2þ gradient. The correction factor is based on three criteria, the ratio of the diVusion coeYcients of the Ca2þ-buVer complex to free Ca2þ, the Ca2þ buVer concentration, and the dissociation constant, Kd, of the Ca2þ buVer compared to the [Ca2þ] of the medium. The Kd is the inverse of the more commonly used ‘‘stability constant’’ also known as the association constant. Only Ca2þ buVers/binding agents that have Kd values near the range of the extracellular [Ca2þ] will act as eVective shuttle buVers during self-referencing of CaSMs. Table III lists a few of these compounds. Note that two of the compounds, ADA and Bicine, are commonly used as Hþ buVers. Generally, the Ca2þ Kd values of other Good buVers are not in the range of normal extracellular [Ca2þ] or are very poor Ca2þ chelators (Dawson et al., 1986). 2þ
E. Measurement of Voltage Gradients The use of CaSMs with self-referencing is subject to a similar problem that occurs with the use of intracellular CaSMs; specifically they detect not only changes in ion concentration but also voltage. Extracellular voltage gradients
4. Electrochemical Measurement of Ca2+ Flux
105
Table III List of Ca2þ binding compounds that can act as shuttle buVers for extracellular Ca2þ in the 0.1–1.0 mM range Ligand
log(Kd)
Pyrophosphate N-(2-acetamido)iminodiacetic acid (ADA) ATP Citric acid Oxalic acid Polyphosphate N,N-bis(hydroxyethyl)-glycine (Bicine)
5.0 4.01 3.8 3.5 3.0 3.0 2.8
Values were obtained from Dawson et al. (1986) except as noted 1(Lance et al., 1983).
have been mapped near many diVerent systems (Borgens et al., 1989; Nuccitelli, 1986) Extracellular electric fields generated by cells are generally very small especially in high conductivity media such as animal saline. However, in lower conductivity saline ion transport can give rise to relatively large electric fields > 1 mV/ 10 mm which can be detected with self-referencing microelectrodes. Plants for example, drive transcellular currents through them, as a result of ion transport across single cells or tissues. In low conductivity medium, these currents generate substantial voltage gradients next to the cells, coexisting with the concentration gradients of the transported ions. The diVerential voltage measured by the CaSM will be the sum of the voltage diVerences due to the [Ca2þ] diVerence and the voltage diVerence. For example, a peak voltage diVerence during oscillating current influx of about 9 mV would occur over a 10-mm distance immediately in front of a lily pollen tube. Peak current density around 0.4 mA/cm2 was measured at a distance of about 20 mm from the cell surface with a medium resistivity of about 5000 O cm (Messerli and Robinson, 1998). This voltage diVerence is just above the background noise of the system used at that time, 5 mV for Ca2þ, (Messerli et al., 1999). The voltage signals detected by the self-referencing CaSM peaked about six times larger than the diVerential voltage due to current flux indicating that the extracellular electric field could have contributed to the calculated Ca2þ flux by up to 15%.
F. Positional Artifacts Self-referencing of CaSMs near solid objects can generate position dependent artifacts. Movement of Ca2þ across the external interface between the liquid membrane and bathing medium may occur through current driven and zero net current mechanisms (Bakker and MeyerhoV, 2000). Release of Ca2þ by the CaSM restricts its sensitivity in bulk medium by leading to a modification of the local ion
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Mark A. Messerli and Peter J. S. Smith
concentration at the tip of the microelectrode. During self-referencing, when the CaSM is positioned near a solid object, released Ca2þ can accumulate between the CaSM and the object in a short period of time leading to an artificially higher concentration of Ca2þ in the constrained space. Likewise, uptake of Ca2þ by the CaSM can lead to a depletion of Ca2þ in the constrained space. These artifacts are most apparent in solutions of low background [Ca2þ]. Figure 3 shows examples of both extremes where CaSMs are self-referenced near a 100-mm diameter glass bead. The CaSM is moving in a path so that the plane of its tip is always parallel to the near surface of the bead to enable the ISM to get closer to the surface. The ion trapping eVect is reduced when the path of excursion orients the plane of the tip of the CaSM perpendicular to the near surface of the solid object, as shown in Fig. 2A and B, because the liquid membrane surface cannot get as close to the solid object. EZux of Ca2þ from the microelectrode tip occurs when constructed with 100 mM CaCl2 backfilling solution, originally performed by Ku¨htreiber and JaVe (1990). Accumulation of the released Ca2þ in less than 1 s can be detected when the bath [Ca2þ] is 50 mM but not when it is 2 mM, giving rise to an artificial eZux of Ca2þ from the solid glass bead. Reducing the concentration of the primary ion in the backfilling solution is one method of reducing the ion leak (Bakker and MeyerhoV, 2000). However, when used with self-referencing this can lead to an artifact of the opposite polarity shown in Fig. 3. The CaSM constructed with
50 100 mM Ca2+ backfill, 50 mM Ca2+ bath 100 mM Ca2+ backfill, 2 mM Ca2+ bath 100 nM Ca2+ backfill, 50 mM Ca2+ bath 100 nM Ca2+ backfill, 2 mM Ca2+ bath 50 mM Ca2+ backfill, 50 mM Ca2+ bath
Differential voltage (mV)
40 30 20 10 0 −10 −20 −30 −40
0
5
10
15
20
25
30
Distance from bead (mm)
Fig. 3 Ca2þ movement across the tip of a CaSM can be detected in low background [Ca2þ] near a solid object. Electroneutral exchange of Ca2þ out of the tip of a CaSM (filled box) or into the tip of the CaSM (filled circle) can give rise to accumulation or depletion of the local [Ca2þ] between a solid object and the tip of the CaSM. In higher bath [Ca2þ] (empty box, empty circle) the accumulation or depletion is insignificant compared to the background [Ca2þ] and is therefore not detected. In lowered bath [Ca2þ] careful balancing of the backfilling [Ca2þ] with the bath [Ca2þ] can reduce (filled triangle) if not eliminate net movement of Ca2þ across the liquid membrane.
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100 nM Ca2þ in the backfilling solution generates an influx of Ca2þ into the CaSM tip, depleting the local [Ca2þ] in the bath and giving rise to an artificial Ca2þ influx into the glass bead. Again this can be detected in the 50 mM Ca2þ solution but not the 2 mM Ca2þ containing bath solution. The artifact can be reduced by matching the backfilling [Ca2þ] with the [Ca2þ] in the bath. Other methods for eliminating Ca2þ flux across the tip of the CaSM include current clamping (Lindner et al., 1999; Pergel et al., 2001), or using the solid contact ion-selective electrode design (Lindner and Gyurcsa´nyi, 2009).
V. Ca2þ Flux Measurements Extracellular Ca2þ flux measurements have been performed on a number of diVerent systems some of which are listed in Table IV, ranging from animal neurons and muscle to tip growing root hairs, pollen tubes, and fungi. Measured Ca2þ fluxes are relatively small ranging between 0.1 and 10 pmol cm 2 s 1 encouraging measurements from cells in reduced background [Ca2þ] 0.1 mM. The limit of flux sensitivity for a typical self-referencing CaSM with 10 mV near real-time variation performed in 1 mM bath [Ca2þ] is about 6.3 pmol cm 2 s 1, an order of magnitude higher than in 0.1 mM bath [Ca2þ]. Considering the large transplasma membrane electrochemical driving force on Ca2þ, reduction of extracellular [Ca2þ] by an order of magnitude did not cause noticeable problems for the diVerent preparations, at least over the few hour period during which measurements were acquired as noted by multiple authors listed in Table IV. While eZux of Ca2þ in cells at rest is expected to be relatively small, the measured influx of Ca2þ, presumably through channels, is also relatively small. Active single 0.5 pS Ca2þ channels at a density of 1 mm 2 should give rise to a Ca2þ influx of about 47 pmol cm 2 s 1. Although as noted by Hille (2001) voltage-gated Ca2þ channels exist at low density and low open probabilities (< 0.1) even with strong depolarizing potentials indicating that low channel density and activity is suYcient to account for measured changes in [Ca2þ]i. The channel density and activity used above may be overestimates of actual Ca2þ channel density. Also, weak influx may also be a result of the Ca2þ amplification cascades that exist to release Ca2þ from intracellular stores after influx through the plasma membrane. Additional directions for the use of self-referencing with CaSMs include the study of electroneutral Ca2þ transporters/exchangers and extracellular Ca2þ signaling (Breitwieser, 2008). Ca2þ selective microelectrodes have been instrumental in providing the sensitivity for defining the complex transport of the Naþ/Ca2þ exchanger (Kang and Hilgemann, 2004) and the P-type plasma membrane Ca2þ pump (PMCA) in neurons (Thomas, 2009). With self-referencing of ion-selective microelectrodes, transport stoichiometries could be determined noninvasively from the outside of intact cells.
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Table IV Calcium flux measurements acquired from preparations representing multiple Kingdoms 2þ
2 1
Preparation
Ca
Aplysia californica bag cell
1 to 5 1 to 5
Rest, H2O2 Rest, thapsigargin
Rana catesbeiana hair cell
0.5 þ5.0 2.5
Rest Stimulated 15% ASWa
4.0
AFWb
0.1
1.0 to 4.0
Rest, Ach.c
0.1
1 to 7.5
Muscarinic agonists Rest, FMRFamide Rest Bepridil additiond Replenished Naþ Addition of EGFe Germination Oscillating tip growth Tip growth, nod factor Tip growth Osmotic regulation Voltage dependence Osmotic regulation
Callinectes sapidus olfaction
Sclerodactyla briareus smooth muscle
Busycon canaliculatum cardiac muscle Mouse ova
flux (pmol cm
s
)
1 to 4
Lilium longiflorum pollen tubes
0.02 þ0.6 0.2 þ0.08 to þ0.35 þ2 to þ20 þ5 to þ38f
Root hairs
þ4.3 to þ7.2 (alfalfa)
Neurospora crassa hyphae
þ2.5 (S. alba) þ0.07 to þ1.2 (A. thaliana) 0.1 þ0.1 to þ1.5
Ceratopteris richardii spores Physcomitrella patens filaments
3.5 top þ0.5 bottom þ1–3
Conditions
Bath [Ca2þ] (mM)
Reference
0.1 None added (0.5 mM EGTA) 0.05
Duthie et al. (1994) Knox et al. (1996)
0.1
0.1
Gleeson et al. (2000b) Gleeson et al. (2000a) Devlin and Smith (1996) Devlin et al. (2003)
0.1
Devlin (1996)
0.05
Pepperell et al. (1999)
None added 0.1 0.13
Hill et al. (1999) Pierson et al. (1994) Messerli et al. (1999)
Not listed
Herrmann and Felle (1995)
0.1 0.1 0.05 0.05
Gravity sensing
0.02–0.05
Gravity sensing
0.1
Yamoah et al. (1998)
Lew (1998) Lew (2007) Lew and Levina (2007) Chatterjee et al. (2000) Allen et al. (2003)
a
Artificial seawater. Artificial freshwater. c Acetylcholine. d Bepridil was added to block the plasma membrane Naþ/Ca2þ exchanger. e Epidermal growth factor. f Calculated flux at cell surface. b
Acknowledgments The BioCurrents Research Center is funded by NIH:NCRR grant P41 RR001395.
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109
References Allen, N. S., Chattaraj, P., Collings, D., and Johannes, E. (2003). Gravisensing: Ionic responses, cytoskeleton and amyloplast behavior. Adv. Space Res. 32(8), 1631–1637. Ammann, D. (1986). Ion-Selective Microelectrodes. Springer-Verlag, Berlin. Ammann, D., Gu¨ggi, M., Pretsch, E., and Simon, W. (1975). Improved calcium ion-selective electrode based on a neutral carrier. Anal. Lett. 8(10), 709–720. Ammann, D., Bu¨hrer, T., Schefer, U., Mu¨ller, M., and Simon, W. (1987). Intracellular neutral carrierbased Ca2þ microelectrode with subnanomolar detection limit. Pflu¨gers Arch. Eur. J. Physiol. 409, 223–228. Bakker, E., and MeyerhoV, M. E. (2000). Ionophore-based membrane electrodes: New analytical concepts and non-classical response mechanisms. Anal. Chim. Acta 416, 121–137. Bakker, E., Meruva, R. K., Pretsch, E., and MeyerhoV, M. E. (1994). Selectivity of polymer membranebased ion-selective electrodes: Self-consistent model describing the potentiometric response in mixed ion solutions of diVerent charge. Anal. Chem. 66, 3021–3030. Bakker, E., Bu¨hlmann, P., and Pretsch, E. (1997). Carrier-based ion-selective electrodes and bulk optodes. 1. General characteristics. Chem. Rev. 97, 3083–3132. Borgens, R. B., Robinson, K. R., Vanable, J. W. J., and McGinnis, M. E. (1989). Electric Fields in Vertebrate Repair. Alan R. Liss, Inc., New York. Breitwieser, G. E. (2008). Extracellular calcium as an integrator of tissue function. Int. J. Biochem. Cell Biol. 40, 1467–1480. Chatterjee, A., Porterfield, D. M., Smith, P. J. S., and Roux, S. J. (2000). Gravity-directed calcium current in germinating spores of Ceratopteris richardii. Planta 210, 607–610. Crank, J. (1967). The Mathematics of DiVusion. Oxford University Press, London. Danuser, G. (1999). Photogrammetric calibration of a stereo light microscope. J. Microsc. 193, 62–83. Dawson, R. M. C., Elliott, D. C., Elliott, W. H., and Jones, K. M. (1986). Data for Biochemical Research. Oxford University Press, Oxford. Devlin, C. L. (1996). A vibrating Ca2þ-selective electrode measures Ca2þ flux induced by the neuropeptide FMRFamide in a gastropod ventricle. Comp. Biochem. Physiol. 116A(2), 93–100. Devlin, C. L., and Smith, P. J. S. (1996). A non-invasive vibrating calcium-selective electrode measures acetycholine-induced calcium flux across the sarcolemma of a smooth muscle. J. Comp. Physiol. 166, 270–277. Devlin, C. L., Amole, W., Anderson, S., and Shea, K. (2003). Muscarinic acetylcholine receptor compounds alter net Ca2þ flux and contractility in an invertebrate smooth muscle. Invert. Neurosci. 5, 9–17. Deyhimi, F., and Coles, J. A. (1982). Rapid silyation of a glass surface: Choice of reagent and eVect of experimental parameters on hydrophobicity. Helv. Chim. Acta 65, 1752–1759. Duthie, G. G., Shipley, A., and Smith, P. J. S. (1994). Use of a vibrating electrode to measure changes in calcium fluxes across the cell membranes of oxidatively challenged Aplysia nerve cells. Free Rad. Res. 20(5), 307–313. Gleeson, R. A., Hammar, K., and Smith, P. J. S. (2000a). Sustaining olfaction at low salinities: Mapping ion flux associated with the olfactory sensilla of the blue crab Callinectes sapidus. J. Exp. Biol. 203, 3145–3152. Gleeson, R. A., McDowell, L. M., Aldrich, H. C., Hammar, K., and Smith, P. J. S. (2000b). Sustaining olfaction at low salinities: Evidence for a paracellular route of ion movement from the hemolymph to the sensillar lymph in the olfactory sensilla of the blue crab Callinectes sapidus. Cell Tissue Res. 301, 423–431. Henriksen, G. H., Raman, D. R., Walker, L. P., and Spanswick, R. M. (1992). Measurement of net fluxes of ammonium and nitrate at the surface of barley roots using ion-selective microelectrodes. Plant Physiol. 99, 734–747. Herrmann, A., and Felle, H. (1995). Tip growth in rooth hair cells of Sinapis alba L.: Significance of internal and external Ca2þ and pH. New Phytol. 129, 523–533.
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Mark A. Messerli and Peter J. S. Smith Hill, J. L., Hammar, K., Smith, P. J. S., and Gross, D. J. (1999). Stage-dependent eVects of epidermal growth factor on Ca2þ eZux in mouse oocytes. Mol. Reprod. Dev. 53, 244–253. Hille, B. (2001). Ion Channels of Excitable Membranes. Sinauer Associates, Inc., Sunderland. Kang, T. M., and Hilgemann, D. W. (2004). Multiple transport modes of the cardiac Naþ/Ca2þ exchanger. Nature 427, 544–548. Knox, R. J., Jonas, E. A., Kao, L.-S., Smith, P. J. S., Connor, J. A., and Kaczmarek, L. K. (1996). Ca2þ influx and activation of a cation current are coupled to intracellular Ca2þ release in peptidergic neurons of Aplysia californica. J. Physiol. 494(3), 627–639. Ku¨htreiber, W. M., and JaVe, L. F. (1990). Detection of extracellular calcium gradients with a calciumspecific vibrating electrode. J. Cell Biol. 110, 1565–1573. Lance, E. A., Rhodes, C. W., III, and Nakon, R. (1983). Free metal ion depletion by ‘‘Good’s’’ buVers. Anal. Biochem. 133, 492–501. Lanter, F., Steiner, R. A., Ammann, D., and Simon, W. (1982). Critical evaluation of the applicability of neutral carrier-based calcium selective microelectrodes. Anal. Chim. Acta 135, 51–59. Lew, R. R. (1998). Immediate and steady state extracellular ionic fluxes of growing Arabidopsis thaliana root hairs under hyperosmotic and hypoosmotic conditions. Physiol. Plant. 104, 397–404. Lew, R. R. (2007). Ionic currents and ion fluxes in Neurospora crassa hyphae. J. Exp. Bot. 58(12), 3475–3481. Lew, R. R., and Levina, N. N. (2007). Turgor regulation in the osmosensitive cut mutant of Neurospora crassa. Microbiology 153, 1530–1537. Lindner, E., and Gyurcsa´nyi, R. E. (2009). Quality control criteriz for solid-contact, solvent polymeric membrane ion-selective electrodes. J. Solid State Electrochem. 13, 51–68. Lindner, E., Gyurcsa´nyi, R. E., and Buck, R. P. (1999). Tailored transport through ion-selective membranes for improved detection limits and selectivity coeYcients. Electroanalysis 11, 695–702. Messerli, M. A., and Robinson, K. R. (1998). Cytoplasmic acidification and current influx follow growth pulses of Lilium longiflorum pollen tubes. Plant J. 16, 87–91. Messerli, M. A., Danuser, G., and Robinson, K. R. (1999). Pulsatile influxes of Hþ, Kþ and Ca2þ lag growth pulses of Lilium longiflorum pollen tubes. J. Cell Sci. 112, 1497–1509. Messerli, M. A., Robinson, K. R., and Smith, P. J. S. (2006). Electrochemical sensor applications to the study of molecular physiology and analyte flux in plants. In ‘‘Plant Electrophysiology,’’ (A. G. Volkov, ed.), pp. 73–107. Springer, Berlin, Heidelberg, New York. Messerli, M. A., Collis, L. P., and Smith, P. J. S. (2009). Ion trapping with fast response, ion-selective microelectrodes enhances detection of extracellular ion channel gradients. Biophys. J. 96, 1597–1605. Munoz, J.-L., Deyhimi, F., and Coles, J. A. (1983). Silanization of glass in the making of ion-sensitive microelectrodes. J. Neurosci. Methods 8, 231–247. Nuccitelli, R. (ed.) (1986). Ionic currents in development. Progress in Clinical and Biological Research. Alan R. Liss, Inc, New York. Pepperell, J. R., Kommineni, K., Buradagunta, S., Smith, P. J. S., and Keefe, D. L. (1999). Transmembrane regulation of intracellular calcium by a plasma membrane sodium/calcium exchanger in mouse ova. Biol. Reprod. 60, 1137–1143. Pergel, E., Gyurcsa´nyi, R. E., Toth, K., and Lindner, E. (2001). Picomolar detection limits with currentpolarized Pb2þ ion-selective membranes. Anal. Chem. 22, 509–514. Pierson, E. S., Miller, D. D., Callaham, D. A., Shipley, A., Rivers, B. A., Cresti, M., and Hepler, P. K. (1994). Pollen tube growth is coupled to the extracellular calcium ion flux and the intracellular calcium gradient: EVect of BAPTA-type buVers and hypertonic media. Plant Cell 6, 1815–1828. Smith, P. J. S., Sanger, R. H., and Messerli, M. A. (2007). Principles, development and applications of self-referencing electrochemical microelectrodes to the determination of fluxes at cell membranes. In ‘‘Electrochemical Methods for Neuroscience,’’ (A. C. Michael, and L. M. Borland, eds.), pp. 373–405. CRC Press, Boca Raton. Speksnijder, J. E., Miller, A. L., Weisenseel, M. H., Chen, T. H., and JaVe, L. F. (1989). Calcium buVer injections block fucoid egg development by facilitating calcium diVusion. Proc. Natl. Acad. Sci. USA 86, 6607–6611.
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Thomas, R. C. (2009). The plasma membrane calcium ATPase (PMCA) of neurones is electroneutral and exchanges 2 Hþ for each Ca2þ or Ba2þ ion extruded. J. Physiol. 587, 315–327. Tsien, R. Y., and Rink, T. J. (1981). Ca2þ-selective electrodes: A novel PVC-gelled neutral carrier mixture compared with other currently available sensors. J. Neurosci. Methods 4, 73–86. Yamoah, E. N., Lumpkin, E. A., Dumont, R. A., Smith, P. J. S., Hudspeth, A. J., and Gillespie, P. G. (1998). Plasma membrane Ca2þ-ATPase extrudes Ca2þ from hair cell stereocilia. J. Neurosci. 18(2), 610–624.
CHAPTER 5
Practical Aspects of Measuring Intracellular Calcium Signals with Fluorescent Indicators Joseph P. Y. Kao, Gong Li, and Darryl A. Auston Center for Biomedical Engineering and Technology, and Department of Physiology University of Maryland School of Medicine Baltimore, Maryland, USA
Abstract I. Introduction II. Fluorescent Ca2þ Indicators III. Loading Indicators into Cells A. Limited Aqueous Solubility of AM Esters B. Dye Compartmentalization: Loading of Indicator into Subcellular Compartments Other than the Cytosol C. Dye Leakage or Extrusion from Cells D. Procedure for Loading IV. Manipulation of [Ca2þ] A. Using EGTA and BAPTA as Extracellular Ca2þ BuVers B. Lowering Extracellular [Ca2þ] C. Divalent Cation Ionophores D. BuVering Changes in Intracellular [Ca2þ] V. Conversion of Indicator Fluorescence Signal into Values of [Ca2þ] A. Calibrating a Nonratiometric Fluorescent Indicator B. Calibrating a Ratiometric Fluorescent Indicator VI. Reporting Indicator Fluorescence Intensity Changes without Calibration A. Reporting Relative Changes in Fluorescence: F/F0 and DF/F0 B. Caveat in Interpreting Relative Fluorescence Changes: Indicator Fluorescence is Not a Linear Function of [Ca2þ] VII. Measuring [Ca2þ] in Mitochondria A. Estimating the Fraction of Intracellular Rhod-2 Indicator that Resides in Mitochondria B. Minimizing Rhod-2 Loading in the Cytosol C. Monitoring Cytosolic and Mitochondrial [Ca2þ] Simultaneously METHODS IN CELL BIOLOGY, VOL. 99 Copyright 2010, Elsevier Inc. All rights reserved.
113
0091-679X/10 $35.00 DOI: 10.1016/S0091-679X(10)99005-5
Joseph P. Y. Kao et al.
114 VIII. Concluding Remarks References
Abstract The use of fluorescent indicators for monitoring calcium (Ca2þ) signals and for measuring Ca2þ concentration ([Ca2þ]) in living cells is described. The following topics are covered in detail: (1) ratiometric and nonratiometric fluorescent indicators and the principles underlying their use, (2) techniques for loading Ca2þ indicators and Ca2þ buffers into living cells, (3) calibration of indicator fluorescence intensity measurements to yield values of intracellular [Ca2þ], (4) analysis of nonratiometric fluorescence intensity data and caveats relating to their interpretation, (5) techniques for manipulating intracellular and extracellular [Ca2þ], and (6) the use of fluorescent indicators to monitor Ca2þ signals in mitochondria. The chapter aims to present these fundamental topics in a manner that is practically useful and intuitively accessible. The origins of key mathematical equations used in the article are outlined in two appendices.
I. Introduction In the application of any measurement technique, a body of practical knowledge is shared by experienced practitioners. Although important for making successful measurements, such lore, which sometimes seems arcane, often is not described explicitly or explained in journal publications. In this respect, measuring [Ca2þ]1 with fluorescent indicators is no exception. The purpose of this chapter is to gather in one place some of the most common and useful practical information relevant to the use of fluorescent Ca2þ indicators. Such a collection of information is hoped to alleviate the frustration of those who are novices at using fluorescent indicators.
II. Fluorescent Ca2þ Indicators The commonly available fluorescent indicators for Ca2þ fall into two operational classes: dual-wavelength ratiometric dyes and single-wavelength nonratiometric dyes (Table I). Chemical structures of some of the indicators listed in Table I are shown in Fig. 1. For nonratiometric indicators, a change in [Ca2þ] brings about a corresponding change in the intensity of the indicator’s fluorescence excitation and emission spectra,2 whereas the wavelengths of the excitation and emission spectral 1 Symbols used: Ca2þ, free calcium ion; [Ca2þ], concentration of free calcium ions; [Ca2þ]i, cytosolic concentration of free calcium ions. 2 An excitation spectrum is taken by monitoring fluorescence emission intensity at a fixed wavelength while excitation light is scanned through a wavelength range over which the sample can absorb light. The emission intensity is plotted as a function of the excitation wavelength. To collect an emission spectrum, excitation light at a fixed wavelength is delivered to the sample while the emission intensity is monitored over a wavelength range. Here, the emission intensity is plotted as a function of emission wavelength.
5. Measuring [Ca2þ] with Fluorescent Indicators
115
Table I Properties of common fluorescent Ca2þ indicatorsa Absorption maxima (nm) Indicator type Nonratiometric Monomeric Quin2 Fluo-2/Fluo-8c Fluo-3 Fluo-4 Calcium Green-1TM Calcium Green-2TM Calcium Green-5NTM Oregon Green 488 BAPTA-1TM Oregon Green 488 BAPTA-2TM Rhod-2 Calcium OrangeTM Calcium CrimsonTM Dextran-conjugatede Fluo-4 dextran (MW 10,000) Calcium Green-1 dextran (MW 3000–70,000)f Oregon Green 488 BAPTA-1 dextran (MW 10,000) Ratiometric Monomeric Fura-2 Fura RedTM Indo-1 Dextran-conjugatede Fura(-2) dextran (MW 10,000)
Emission maxima (nm)
Kd (nM)
Ca -free Ca -bound Ca2þ-free Ca2þ-bound
115b 380 400 345 190 550 14d 170 580 1.0d 328 185
352 – 503 491 506 506 506 494 494 556 549 589
332 492 506 494 506 503 506 494 494 553 549 589
492 – 526 516 531 536 532 523 523 576 575 615
498 514 526 516 531 536 532 523 523 576 576 615
600 240–540f 265
494 509 496
494 509 496
518 534 524
518 534 524
224b 140 250b
362 473 349
335 436 331
512 670 485
505 655 410
240
364
338
501
494
2þ
2þ
a
Data from Tsien (1980), Grynkiewicz et al. (1985), Minta et al. (1989), Haugland (1992), and Molecular Probes, The Handbook (web publication, Invitrogen Corporation). b EVective Kd in the presence of 1 mM Mg2þ. (Generally, competition by Mg2þ slightly reduces the aYnity of any indicator for Ca2þ.) c DiVerent names for the same molecule. d mM. e The Kd and absorption and emission maxima of dextran-conjugated indicators can vary from lot-to-lot and is dependent on the molecular weight of the dextran used as well. f Kd is reported to be diVerent between low- and high-MW versions: 540 nM for MW 3000, 240–250 nM for MW 10,000 and 70,000.
peaks remain essentially unchanged. Excitation spectra of Fluo-3 (Minta et al., 1989), a nonratiometric indicator, at saturating and ‘‘zero’’ [Ca2þ] are shown in Fig. 2. It can be seen that peak excitation occurs at 505 nm irrespective of whether the indicator is Ca2þ-free or Ca2þ-bound—the defining characteristic of a nonratiometric indicator. In contrast, ratiometric indicators exhibit not only intensity changes with changing [Ca2þ] but the Ca2þ-free and Ca2þ-bound forms
Joseph P. Y. Kao et al.
116 CO2−
−
CO2− −O2C
CO2−
O2C
N O
O2C
N
N
− O2C CO2− CO2− −O2C N N
−
CO2− −O2C
N O
O
O
O
EGTA
N
O
Quin2
BAPTA CO2−
−
CO2− −O2C
O 2C
CO2− −O2C
N
N O
O
−
CO2−
O 2C N
N
O
O
O
N O
NH
Fura-2
CO2− CO2−
−
CO2− −O2C
O2C
N
CO2−
CO2−
N
X
O
O
O
X
R
2 or −8*
H
H or CH3
3
Cl
CH3
4
F
Fluo-
O−
CO2−
O2C N
O
N+
R X
−
CO2− −O2C
N
O O
Indo-1
Rhod-2
O N
CH3 −
CO2− −O2C
O2C N
N
X O
O
O
O N H
N H
Calcium green-2 Oregon green 488 BAPTA-2
X
Cl F
X CO2−
O O
X O−
−
O2C
O O
X −
O
Fig. 1 Structures of selected fluorescent Ca2þ indicators and the Ca2þ chelators, EGTA, and BAPTA. All molecules are represented in their polycarboxylate, Ca2þ-sensitive forms. The following conventions have been used in these structural drawings: (1) Implicit carbon: Every unlabeled vertex, whether internal or terminal, represents a carbon atom. (2) Implicit hydrogen: Every carbon has a suYcient number of (undrawn) hydrogens to make the total number of bonds to that carbon equal to 4. (3) Explicit heteroatoms: non-carbon, non-hydrogen atoms (e.g., O, N) are labeled explicitly; hydrogens OH is equivalent to attached to the heteroatom are also explicitly drawn. For example, CH3–CH¼CH–CH2–OH. *DiVerent names for the same molecule.
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Fluorescence intensity
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420
440
460
480
500
520
Wavelength (nm)
Fig. 2 Excitation spectra for Fluo-3 (lemission ¼ 525 nm). The Ca2þ-free form of Fluo-3 is 100 times less bright than the Ca2þ-bound form.
Fluorescence intensity
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300
350 400 Wavelength (nm)
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Fig. 3 Excitation spectra of Ca2þ-bound and Ca2þ-free forms of Fura-2 (lemission ¼ 505 nm).
of the indicator actually have distinct spectra, the maxima in which occur at diVerent wavelengths (the spectra show wavelength shifts). The two ratiometric indicators most commonly used are Fura-2 and Indo-1 (Grynkiewicz et al., 1985). For Fura-2, significant shifts are observed in the excitation spectra (Fig. 3) but not in the emission spectra. Indo-1 shows a significant shift primarily in its emission spectra. For nonratiometric indicators, because intensity monitored at a single-
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wavelength is the only experimental measurement that is related to [Ca2þ], intensity changes arising from factors unrelated to changes in [Ca2þ] (e.g., changes in cell thickness, leakage of indicator from the cell) can confound interpretation of the intensity data. In contrast, because the Ca2þ-free and Ca2þ-bound forms of ratiometric indicators are characterized by spectral peaks at diVerent wavelengths, intensity measurements can be made at two diVerent wavelengths, and the ratio between these intensities is quantitatively related to [Ca2þ] (Grynkiewicz et al., 1985). Obtaining a ratio minimizes the eVect of many artifacts that are unrelated to changes in [Ca2þ]—for example, a change in cell thickness or indicator loss from the cell would aVect intensities at the two wavelengths equally, so the eVect would cancel when the two intensities are ratioed. The two commonly used ratiometric indicators, Fura-2 and Indo-1, require excitation in the ultraviolet (UV) range, whereas most of the common nonratiometric dyes use visible excitation light. Although the ratiometric dyes can be calibrated more reliably (Section V), sometimes avoiding using UV light for excitation may be necessary (e.g., UV can excite significant autofluorescence in some biological preparations and can photolyze photosensitive ‘‘caged’’ compounds). Clearly, in practice, instrumentation for using ratiometric indicators is more complex than that for nonratiometric indicators. Quin2 (Tsien, 1980; Tsien et al., 1982) is the archetypal tetracarboxylate indicator listed in Table I (structure in Fig. 1). Its properties and applications as a nonratiometric indicator have been reviewed in detail (Tsien and Pozzan, 1989). However, Quin2 has been superseded by new generations of nonratiometric and ratiometric indicators. Of the nonratiometric indicators listed in Table I, the Fluo and Calcium Green series as well as Oregon Green 488 BAPTA-2 incorporate fluorescein chromophores and are, therefore, excited at wavelengths typical of fluoresceins. The Fluo dyes, Calcium Green-2 and Oregon Green 488 BAPTA-2 exhibit the largest intensity changes in their transition from Ca2þ-free to Ca2þ-bound forms ( 100fold; Haugland, 1992; Minta et al., 1989). This change can be an advantage because, for a given rise in [Ca2þ], these indicators give a larger increase in brightness compared to other nonratiometric indicators. Because fluorescence quantum eYciency3 can range only from 0 to 1, the large intensity diVerence between Ca2þbound and Ca2þ-free forms implies that the Ca2þ-free forms of the two indicators must be only weakly fluorescent. Some researchers find this fact annoying because cells with relatively low resting [Ca2þ]i (cytosolic free Ca2þ concentration) would have most of the indicator in the Ca2þ-free form and therefore would be quite dim. Rhod-2, Calcium Orange, and Calcium Crimson are indicators that incorporate rhodamine-type chromophores and therefore are excited at much longer wavelengths than are the Fluo and Calcium Green dyes. When the acetoxymethyl (AM) 3 Fluorescence quantum eYciency, symbolized as FF or QF, is the fraction of total light absorbed that is emitted as fluorescence. Fluorescence quantum eYciency may also be thought of as the probability that a molecule will emit fluorescence after absorbing a photon. Being a probability, the quantum eYciency can have a value between 0 and 1.
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ester is used to load cells, Rhod-2 loads well into mitochondria; up to 80% of the intracellular dye is located in these organelles. The use of Rhod-2 to monitor Ca2þ signals in mitochondria is outlined in Section VII. A choice of ratiometric indicator can be made on practical grounds. Typically, Fura-2 is excited alternately at two diVerent wavelengths, whereas the emission is collected at a single fixed wavelength. Therefore, the pair of intensity measurements, whether in imaging or in single-cell microfluorometry, must be collected sequentially. Indo-1, on the other hand, usually is excited at a fixed wavelength whereas emission is monitored simultaneously at two diVerent wavelengths, that is, emission from the Ca2þ-bound and Ca2þ-free forms of the indicator can be collected simultaneously. Therefore, Indo-1 potentially can give better temporal resolution. However, in conventional imaging, Indo-1 can be more diYcult to use because the two emission images, usually collected through slightly diVerent optical paths, can be diYcult to keep in spatial registration. Fura-2 has been the most widely used ratiometric Ca2þ indicator, both in conventional imaging and in single-cell measurements. Indo-1 has, however, been used successfully in UV laserscanning confocal imaging applications (Motoyama et al., 1999; Niggli et al., 1994; Sako et al., 1997). Fura RedTM is touted as a ratiometric indicator whose excitation and emission wavelengths are both in the visible range. This indicator suVers from having very low fluorescence quantum eYciency ( 0.013 in the Ca2þ-free form; J.P.Y. Kao, unpublished results4). Fura Red diVers from the other ratiometric indicators because its fluorescence intensity decreases upon binding Ca2þ. The relatively low quantum eYciency implies that higher indicator concentrations and/or higher excitation light intensities are required. The dextran-conjugated dyes are biopolymers with pendant indicator molecules. The dextran-conjugated indicators listed in Table I are available with dextran molecular weights of 3000, 10,000, or 70,000 (Invitrogen Corporation, Molecular Probes Brand). Being membrane-impermeant, dextran conjugates must be loaded into cells by an invasive technique such as microinjection. Whereas the monomeric indicators can leak out of cells at a steady rate (Section III.C), dextran-conjugated indicators tend to have long residence times in cells. Therefore, dextran-conjugated dyes can be useful in applications in which long-term monitoring of [Ca2þ]i is required. Instances also occur in which cells rapidly transport monomeric dyes into internal organelles (Hepler and Callaham, 1987) but do not do so when dextran conjugates are used. Because the conjugates are made by covalent attachment of monomeric indicators to dextran polymers, individual indicator monomers can reside in slightly diVerent local microenvironments on the polymer. Therefore, the conjugates, rather than having a unique Kd and identical spectral properties, are characterized by a range of microscopic Kds and a distribution of spectral properties. These characteristics provide a likely explanation for lot-to-lot variations in Kd and spectral characteristics. 4 The quantum eYciency of the Ca2þ-free form of Fura Red was determined relative to carboxySNARF-1.
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120 Membrane-permeant AMO2C CO2AM CO2AM AMO2C Indicator Ca2+-insensitive
Ca2+-sensitive AMO2C CO2AM CO2AM AMO2C Indicator
−
Esterases
−
O2C CO2−
O 2C
CO−2
Indicator Trapped inside cell
Extracellular
Intracellular
Fig. 4 Schematic representation of how incubation with the acetoxymethyl (AM) ester results in intracellular accumulation of a polycarboxylate indicator. The hydrophobic (lipophilic) AM ester readily diVuses into the cell through the cell membrane. Abundant cellular esterases cleave the AM ester groups to generate the Ca2þ-sensitive form of the indicator which, being a polyanion, cannot escape through the cell membrane and is, therefore, trapped inside the cell.
III. Loading Indicators into Cells The common fluorescent indicators for Ca2þ are polycarboxylate anions that cannot cross lipid bilayer membranes and therefore are not cell-permeant. In the negatively charged form, the indicators can be introduced into cells only by microinjection or through transient cell permeabilization, procedures that require some special equipment and skill.5 By far the most convenient way of loading an indicator into cells is incubating the cells in a dilute solution or dispersion of the AM ester of the indicator. This process is represented schematically in Fig. 4. The AM group is used to mask the negative charges on the carboxyl groups present in the indicator molecule. The AM ester form of the indicator is uncharged and hydrophobic. Consequently, it can pass through the cell membrane and enter the cell interior. The carboxyl groups in the indicator, however, are essential to the ability of the indicator molecule to sense Ca2þ; therefore, the AM groups must be removed once the AM ester has entered the cell. Because the AM group is labile to enzymatic hydrolysis by esterases present in the cell, the AM esters are processed intracellularly to liberate the Ca2þ-sensitive polycarboxylate form which, being multiply charged, becomes trapped inside the cell. Trapping of the polyanionic form of the indicator allows cells to accumulate up to hundreds of micromolar of
5 A variety of techniques for loading membrane-impermeant species into cells is discussed by McNeil (1989, 2001).
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the Ca2þ-sensitive form of the indicator when incubated with micromolar concentrations of the AM ester in the extracellular medium. Several factors influence the eYciency and quality of indicator loading via the AM ester, and will be discussed subsequently.
A. Limited Aqueous Solubility of AM Esters AM esters of the common Ca2þ indicators have molecular weights in excess of 1000. Being large uncharged organic molecules, these esters have very low solubility in aqueous media. For example, at 25 C, the solubility of the AM ester of Fura2 in pure water is only 0.11 mM (Kao et al., 1990). In biological media, in which the ionic strength is typically 0.15 M, the solubility of Fura-2 AM would be even lower. Addition of AM ester in excess of the solubility limit simply would result in precipitation of solid AM ester, which is eVectively unavailable for loading cells. In addition, fine particles of solid AM ester often adhere well to the outer surfaces of cells or to the extracellular matrix and can contribute large Ca2þ-insensitive fluorescence signals to the measurement.6 A convenient solution to the solubility problem is the use of Pluronic F-127, a mild nonionic surfactant,7 as a dispersing agent for AM esters. Typically, aliquots of Pluronic and AM ester stock solutions in dimethylsulfoxide (DMSO) are mixed intimately before dispersal into an aqueous medium.8 The Pluronic is presumed to sequester the AM ester in micellar form, thus preventing precipitation, and the micelles serve as a steady source to replenish AM esters taken up by cells. The net result is significantly improved loading of indicators into cells. Details of the loading procedure are described in Section III.D below.
B. Dye Compartmentalization: Loading of Indicator into Subcellular Compartments Other than the Cytosol
1. Minimizing Compartmentalization In typical experiments, one usually wishes to monitor changes in the concentration of Ca2þ in the cytosol; therefore, ideally, one would like the indicator to be loaded exclusively into the cytosol. This ideal situation is almost never realized for two reasons. First, because the AM ester form of the indicator is membrane-permeant, it can enter not only the cytosol but all subcellular 6
This is a problem with AM ester that are fluorescent (e.g., Fura-2 AM and Indo-1 AM) but not with nonfluorescent AM esters (e.g., AM ester of the Fluo series). 7 Pluronic F-127 is manufactured by BASF Wyandotte Corporation, Wyandotte, Wisconsin. Being a surfactant used on the industrial scale, it is inexpensive. 8 With slight warming, Pluronic F-127 can be dissolved in DMSO at almost 25% (w/v). Such a highly concentrated stock solution is inconvenient to use, however: as the solution absorbs moisture from the air, the Pluronic will precipitate, and the resulting particulate suspension is diYcult to pipette. A 15–20% solution in DMSO is a convenient formulation.
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membrane-enclosed compartments as well. Although this process occurs to a large extent in the cytosol, enzymatic hydrolysis of AM esters also can take place within subcellular organelles. Therefore, some fraction of the indicator molecules tend to be trapped in noncytosolic compartments. Second, some cell types actively endocytose material from the incubation medium (Malgaroli et al., 1987), including dispersed AM esters, which are then hydrolyzed to release fluorescent indicator molecules within organelles of the endocytotic pathway. Presumably, indicator molecules liberated in this way can end up in a variety of organelles that are connected to the endocytotic pathway by vesicular traYc. Because most subcellular organelles [e.g., endoplasmic reticulum (ER), lysosomes] tend to have high intraorganellar [Ca2þ] (> mM), indicators confined to these organelles would be saturated with Ca2þ and would contribute a high-[Ca2þ] fluorescence signal that would not vary with changes in cytosolic [Ca2þ]. Therefore, the net eVect of compartmentalized dye is biasing measured cytosolic free Ca2þ concentration toward higher values. The first cause of dye compartmentalization just stated is a reflection of an inherent imperfection of the AM ester loading technique and cannot be remedied easily. The second cause is a cell biological process and can be attenuated. Because endocytosis is a temperature-dependent process, cells loaded at lower temperatures with AM ester tend to show less compartmentalization of indicator (Malgaroli et al., 1987). The following results illustrate this point. In REF52 fibroblast cells, roughly 30% of total intracellular Fura-2 was in noncytosolic compartments when loading via the AM ester was carried out at 37 C, whereas only 10% was compartmentalized when loading was performed at 23 C. Although endocytosis is known to be blocked at 10 C in mammalian cells and at 4 C in amphibian cells, loading at the lowest biologically permissible temperature does not necessarily yield the best results, because processing of AM esters by esterases in the cytosol is also temperature-dependent. At very low temperatures, the concentration of indicator accumulated in the cytosol can be quite low. Optimal loading temperature is determined empirically to be the temperature at which dye compartmentalization is minimized while good cytosolic loading is maintained. In practice, convenience often dictates loading at room temperature as a reasonable compromise.9
2. Assessing Extent of Compartmentalization The extent of indicator compartmentalization can be estimated through a simple semiquantitative procedure that is based on the observation that micromolar concentrations of digitonin primarily permeabilizes the plasma membrane 9 When loading is done in air, the incubation medium should be bicarbonate-free (i.e., some other buVer such as HEPES should be used to maintain the pH of the medium). Otherwise, steady loss of CO2 will shift the CO2–HCO3–CO32 equilibrium and rapidly alkalinize the medium.
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Fluorescence intensity (a.u.)
Digitonin
Fi Triton X-100
Fd Fb
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200 Time (s)
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Fig. 5 Procedure for assessing dye compartmentalization. REF52 fibroblast incubated with 1 mM Fura-2 AM dispersed with Pluronic F-127 in Minimal Essential Medium (MEM) for 60 min in an air incubator at 37 C. Measurement was done in Hanks’ Balanced Salt Solution (HBSS) containing 2.6 mM EGTA (suYcient to reduce extracellular [Ca2þ] to <1 mM), and buVered at pH 7.4 with HEPES. The concentration of digitonin was 20 mM; that of Triton X-100, 1%, w/v. (a.u. = arbitrary units).
and release cytosolic dye, whereas 1% Triton X-100 can permeabilize and release dye from subcellular organelles (e.g., see Kao et al., 1989). The procedure consists of monitoring total fluorescence from a cell or a field of cells, preferably bathed in low-Ca2þ medium,10 and treating the cells first with digitonin and then with Triton X-100. Figure 5 shows the procedure being applied to a cell loaded with Fura-2 AM. The fluorescence measured before treatment (Fi) represents contributions from cytosolic and compartmentalized dye plus background. After digitonin release of cytosolic dye, the fluorescence measured (Fd) represents compartmentalized dye plus background. The fluorescence level attained after Triton X-100 treatment is considered the background (Fb). The fraction of total intracellular dye that is compartmentalized in organelles is simply (Fd Fb)/ (Fi Fb).
10 Nominally Ca2þ-free medium (i.e., medium from which Ca2þ salts have been omitted) or, better still, medium containing suYcient EGTA to make [Ca2þ] < 1 mM (see Section IV.B.1). It is important to keep [Ca2þ] low in this experiment because when [Ca2þ] is elevated, permeabilized cells lose their integrity rapidly, and dye molecules may leak from organelles even before Triton is applied.
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B 1.0
1.0
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0.9 Fraction remaining
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0.8
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0.8 Proben. Sulfin. 0.7
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No inhibitor 0.6
0.5 0
10
20 Time (min)
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0
5
10 15 Time (min)
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Fig. 6 (A) Time course of indicator loss at 30 and 35 C from REF52 cells loaded with Fura-2. (B) EVect of probenecid (1 mM) and sulfinpyrazone (75 mM) on indicator extrusion from REF52 cells at 35 C; arrow marks the time of drug application. For all measurements, the cells were loaded with Fura2 by incubation with AM ester/Pluronic dispersion in HEPES-buVered HBSS at 25 C. Experiments were done in HBSS at the specified temperatures. Total Fura-2 fluorescence (FT ¼ F340 þ F380) is monitored over time. Each trace was normalized by dividing by the value of FT at time 0.
C. Dye Leakage or Extrusion from Cells Once an indicator is loaded into cells, it leaks out at a rate that is strongly temperature-dependent. For mammalian cells, the loss rate is maximal at 37 C and drops oV sharply as temperature is lowered,11 as shown in Fig. 6A. Loss of indicator occurs by an extrusion mechanism for organic anions (Di Virgilio et al., 1988) and can be blocked eVectively by inhibitors of uric acid transport such as probenecid and sulfinpyrazone (Di Virgilio et al., 1988, 1990), as illustrated in Fig. 6B. Sulfinpyrazone at tens to hundreds of micromolar has been found to be eVective, whereas probenecid works at millimolar dosage. High concentrations of inhibitor virtually can stop indicator extrusion but also can induce signs of cellular stress such as blebbing. Therefore, using the minimum concentration of inhibitor necessary to reduce the rate of indicator loss to a tolerable level without attempting to block the process altogether is advisable. In general, slowing indicator loss by lowering the temperature at which experiments are conducted is preferable to using transport inhibitors.
11 The sharp increase in rate of indicator leakage with rising temperature is another reason why compartmentalization is a more severe problem when cells are incubated with AM esters at higher temperatures. Higher temperature accelerates loss of dye from the cytosol but not from organelles, so compartmentalized dye becomes a larger proportion of the total intracellular dye content.
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Probenecid and sulfinpyrazone are both hydrophobic organic acids and, as such, are practically insoluble in water. These molecules must be neutralized with a stoichiometric amount of base (e.g., NaOH) before an aqueous stock solution can be prepared.
D. Procedure for Loading Stock solutions of the AM ester of the indicator can be made with dry DMSO as solvent. Typical concentrations can be in the range of 0.1–10 mM. Such DMSO solutions may be stored safely in screw-capped polypropylene microcentrifuge tubes in the freezer for many months without apparent degradation. Dry DMSO is used for making a solution of Pluronic F-127 at a concentration of 15–20% (w/w or w/v is not crucial). This stock solution can be stored at room temperature. When exposed to air, this concentrated stock solution slowly absorbs moisture until Pluronic F-127 begins to precipitate. Because a heterogeneous Pluronic stock is diYcult to transfer, preparing a fresh stock is preferable at this stage. Usually one can load cells by incubation in medium containing (nominally) 0.1 to a few tens of micromolar AM ester in a Pluronic dispersion. Typically 0.5–1 mL of Pluronic stock is suYcient for dispersing 1–10 nmol AM ester. Thus, 1 mL of 20% Pluronic stock is adequate for dispersing 1 mL of 10 mM AM ester stock into 1 mL of medium to yield a 10 mM AM ester solution (1000-fold dilution). Using a dilution of 1:1000 minimizes the DMSO concentration in the final loading medium (to a few parts per thousand). Premixing the requisite volumes of AM ester stock and Pluronic stock is advisable since this minimizes the chances of AM ester precipitation during aqueous dispersal. The Pluronic-AM mixture is then dispersed into aqueous loading medium. Serum proteins [e.g., bovine serum albumin (BSA)] often have a salutary eVect on cells and also can improve loading eYciency, possibly by acting as hydrophobic carriers for the AM ester and preventing its precipitation. Thus, including BSA (0.5–1%) or serum (a small percentage) in the incubation medium may be advantageous. Enzymatic processing of an AM species to yield the Ca2þ-sensitive indicator typically consists of sequential hydrolysis of four to eight AM ester groups, depending on the particular indicator chosen. Only when all the AM groups on a molecule are hydrolyzed does the molecule become properly Ca2þ-sensitive. For this reason, after removal of AM-containing loading medium, allowing the cells some extra time (e.g., 20 min at room temperature) to complete intracellular processing of the most recently trapped, partially hydrolyzed AM species can be useful in some cases. It is reassuring to convince oneself that suYcient AM ester is present in the loading medium, so the amount of indicator taken up by cells is not limited by the amount available. For a typical example, assume that 1 million cells are loaded in 2 mL medium containing 2 mM AM ester. The total amount of AM ester available
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is 4 nmol. If the cells are 20 mm in diameter, then the 1 million cells have a total intracellular volume of 4.2 mL. Further, if cells are loaded to a final intracellular indicator concentration of 150 mM (a generous estimate), then the total amount of AM uptake by cells is 0.63 nmol, which is still much less than the 4 nmol available in the loading medium.
IV. Manipulation of [Ca2þ] In studying Ca2þ-dependent cellular processes, raising or lowering intracellular or extracellular [Ca2þ] is frequently desirable. Conventional techniques for achieving these ends require the use of Ca2þ buVers or ionophores and will be discussed in this section.
A. Using EGTA and BAPTA as Extracellular Ca2þ BuVers Because it is highly selective for binding Ca2þ over Mg2þ,12 EGTA is the most commonly used Ca2þ buVer. However, because two of the ligand atoms in EGTA are tertiary alkylamino nitrogens, the two highest pKas of EGTA are 8.90 and 9.52,13 implying that at physiological pH EGTA will exist primarily as protonated species—a fact that is illustrated more quantitatively in Fig. 7. For example, Fig. 7 shows that, at pH 7.2, 98% of EGTA in solution exists as H2EGTA2, 2% as HEGTA3, and only a negligible fraction is in the EGTA4 form. Therefore, the Ca2þ-binding reaction near physiological pH is fairly represented as H2 EGTA2 þ Ca2þ > CaEGTA2 þ 2Hþ That two Hþ ions are liberated in the binding reaction means that the binding of Ca2þ by EGTA should have very steep pH dependence, as a plot of pK0 d(Ca)14 versus pH indeed shows (Fig. 8). For a concrete example, a drop in pH from 7.2 to 7.1 changes the K0 d(Ca) of EGTA by a factor of 1.6, that is, small errors in pH can lead to significant uncertainties in the dissociation constant. In contrast, For EGTA, DpKd ¼ pKd(Ca2þ) pKd(Mg2þ) ¼ 5.58; therefore, EGTA binds Ca2þ more tightly than Mg by a factor of 380,000 (i.e., 105.58). For comparison, in the case of EDTA, DpKd ¼ 1.78, which represents only a 60-fold diVerence in EDTA’s aYnity for Ca2þ and Mg2þ. BAPTA [1,2-bis(o-aminophenoxy)ethane-N,N,N0 ,N0 -tetraacetic acid] has a selectivity similar to that of EGTA: DpKd ¼ 5.20. 13 At 25 C and 0.10 M ionic strength. Data pertaining to EGTA that are used in this section are from Martell and Smith (1974). 14 In the metal chelator literature, Kd is used for the ‘‘absolute’’ (or intrinsic) dissociation constant and represents the dissociation constant characterizing the fully deprotonated form of the chelator. K0 d represents Kd that has been corrected for the weakening eVect of acidic pH (thus K0 d is the working dissociation constant at a specific pH). This convention (Kd vs. K0 d) is not followed consistently in the applications literature. Details of how pH correction is applied to convert Kd into K0 d are described in Appendix 1. 12
2þ
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Free EGTA existing as H2EGTA2−, HEGTA3−, or EGTA4− (%)
100
EGTA4−
H2EGTA2−
50
HEGTA3−
0 6
7
8
9 pH
10
11
12
Fig. 7 Percentage of free EGTA existing as H2EGTA2, HEGTA3, and EGTA4 as a function of solution pH. Calculations performed with data for EGTA (at 0.1 M ionic strength, 25 C) tabulated by Martell and Smith (1974); see Appendix 1 for algebraic details.
knowing that the two highest pKas of BAPTA are 5.47 and 6.36 (Tsien, 1980), one infers that the ability of BAPTA to bind Ca2þ should be only very weakly dependent on pH, as shown in Fig. 8. Comparison of the two traces in Fig. 8 shows that BAPTA has the advantage of being only weakly pH dependent in the physiological pH range. The fact that the two traces cross between pH 7.2 and 7.3 implies that EGTA has the potential advantage of being a progressively stronger binder of Ca2þ above the crossover point (e.g., about two- and ninefold stronger than BAPTA at pH 7.5 and 7.8, respectively). The pH insensitivity of BAPTA makes it a less troublesome Ca2þ buVer to use, although it is more costly than EGTA.
B. Lowering Extracellular [Ca2þ] In an experiment, lowering extracellular [Ca2þ] is often desirable. Depending on how low one wishes to clamp the extracellular [Ca2þ], one of the approaches described in the following sections may be adopted. The procedures require either a stock solution of 1 M Na2H2EGTA at a pH near neutral or a stock solution of 1 M Na4BAPTA.
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pK⬘d
9 8 7 BAPTA 6 5 4 6
7
8
9
10
11
12
pH
Fig. 8 Plot of pK0 d(Ca) versus pH for EGTA and BAPTA. Calculations performed with data for EGTA from Martell and Smith (1974) and for BAPTA from Tsien (1980).
1. Lowering [Ca2þ] to <1 mM but not Approaching 0 a. Using EGTA Na2H2EGTA can be added directly to calcium-containing medium at a concentration that is 3–4 times15 the concentration of Ca2þ in the medium. Because binding of Ca2þ to H2EGTA2 releases protons and acidifies the medium, simultaneously adding TRIS base [tris(hydroxylmethyl)aminomethane] at a concentration equal to 2.2 times the Ca2þ concentration in the medium is also necessary. This amount of TRIS scavenges protons released from H2EGTA2 and maintains the pH of the medium at nearly the level before EGTA addition. This procedure can be applied confidently down to pH 6.8. At pH levels that are much lower, the strong pH sensitivity weakens EGTA and makes it inconvenient to use as a Ca2þ scavenger. b. Using BAPTA Add Na4BAPTA at a concentration equal to 3 times the Ca2þ concentration in the medium. Essentially no pH adjustments are necessary. Because of its relative insensitivity to decreasing pH (Fig. 8), BAPTA can be used more conveniently for scavenging Ca2þ at lower pH values than EGTA. 15
3 times if pH 7.0, 4 times if pH < 7.0.
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2. Lowering [Ca2þ] to a Level Approaching 0 If one desires a medium in which the free Ca2þ concentration approaches ‘‘zero,’’ one can prepare nominally calcium-free medium16 that also contains EGTA or BAPTA at millimolar concentrations. Again, to avoid problems of rapid weakening of the Ca2þ aYnity of EGTA with decreasing pH, using BAPTA at pH < 7 is more convenient.
3. Setting Extracellular [Ca2þ] to a Precisely Known Value When the extracellular [Ca2þ] must be known precisely, medium containing a well-defined Ca2þ buVer system at a fixed pH must be made. The preparation of such solutions is detailed in Chapter 9.
C. Divalent Cation Ionophores
1. Properties of Br-A23187 and Ionomycin Br-A2318717 and ionomycin are ionophores that form complexes with divalent metal cations; the complexes are lipid-soluble and thus can cross cellular membranes. These two ionophores are commonly used to increase the permeability of biological membranes to Ca2þ. Understanding the diVerences between the two makes it possible to make a judicious choice during an experiment. Ionomycin can lose two acidic hydrogens and, as a dianion, can form an uncharged 1:1 complex with a divalent metal ion such as Ca2þ or Mg2þ. BrA23187 can lose a single acidic hydrogen and form an uncharged 2:1 complex with a divalent metal ion. This diVerence makes ionomycin potentially more eVective in binding and transporting divalent cations (e.g., two molecules of BrA23187 are needed to bind and carry a single Ca2þ whereas only one molecule of ionomycin is suYcient). Compared with Br-A23187, ionomycin has somewhat better selectivity for Ca2þ over Mg2þ; ionomycin prefers Ca2þ by a factor of 2, whereas Br-A23187 shows essentially no preference for one cation over the other (Liu and Hermann, 1978). In addition, these ionophores actually do not bind Ca2þ very tightly [e.g., Kd(Ca2þ) 100 mM for ionomycin; J.P.Y. Kao, unpublished results.18] These factors suggest that the two ionophores would be ineYcient in mediating Ca2þ transport when relatively low Ca2þ concentrations are involved (i.e., at [Ca2þ] Kd, e.g., < 1 mM), because at such low Ca2þ concentrations, only a minute fraction of total 16 Because calcium is a ubiquitous ‘‘contaminant’’ in the environment, nominally calcium-free solutions still can contain micromolar levels of Ca2þ. 17 The parent compound, A23187, is fluorescent and should be avoided in fluorescence work. The presence of the bromine atom in 4-bromo-A23187 (Br-A23187) eVectively quenches the intrinsic fluorescence of the ionophore and makes the molecule useful in fluorescence microscopy. 18 Determined by absorption spectroscopy at pH 11, at which all ionomycin in solution would be present as the fully deprotonated dianion.
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ionophore is actually engaged in Ca2þ binding and transport. This problem becomes evident when either ionophore is used in calibrating Ca2þ indicators in cells (see Section V.B). The most significant diVerence between the two ionophores lies in the pH dependence of their ability to transport Ca2þ (Liu and Hermann, 1978). Transport of Ca2þ by Br-A23187 approaches a maximum at pH 7.5, whereas Ca2þ transport by ionomycin does not reach a maximum until pH 9.5. The pH at which halfmaximal transport is achieved is 6.4 for Br-A23187 and 8.2 for ionomycin. Therefore, if one desires to increase transport of extracellular Ca2þ into cells in acidic media (pH < 7.0), Br-A23187 is a much better choice than ionomycin.
2. Using Br-A23187 and Ionomycin Ionomycin can be obtained as either the free acid or the Ca2þ salt. Br-A23187 is available as the free acid. All forms are soluble in dry DMSO, which can be used to prepare stock solutions. Because these ionophores are very hydrophobic, they are bound avidly by serum proteins. Serum proteins such as BSA, when present in the medium, greatly reduce the eVectiveness of ionophores and, if possible, should be left out of the experimental medium when ionophores are to be used. Otherwise, much higher concentrations of ionophore must be used. Br-A23187 and ionomycin have been used at concentrations ranging from 10 7 to 10 5 M. In addition to increasing Ca2þ flux across the plasma membrane, Br-A23187 and ionomycin also transport Ca2þ out of intracellular calcium stores into the cytosol. Therefore, in the presence of these ionophores, intracellular calcium stores are rapidly depleted (Kao et al., 1990). D. BuVering Changes in Intracellular [Ca2þ]
1. Increasing Intracellular Ca2þ BuVering Capacity by BAPTA Loading When a change in [Ca2þ]i (a Ca2þ signal) is correlated with a biological process, one can ascertain whether the Ca2þ signal is essential in the process by blocking the change in [Ca2þ]i with a calcium chelator. By far the easiest way to introduce extra Ca2þ buVering capacity into cells is by incubation with BAPTA AM in Pluronic dispersion. Compared with the AM esters of common Ca2þ indicators, BAPTA AM has much higher aqueous solubility—15 mM at 25 C (Kao et al., 1990). Therefore, BAPTA can be loaded eYciently into cells via the AM ester. BAPTA AM is loaded into cells in precisely the same way that AM esters of indicators are loaded. Cells can be loaded with AM esters of BAPTA and an indicator simultaneously. Figure 9 illustrates the eVect of intracellular BAPTA loading on normal changes in [Ca2þ]i. Figure 9A shows the changes in [Ca2þ]i in a REF52 cell loaded with Fura-2 in response to sequential application of 1 mM bradykinin and 1 mM Br-A23187. Figure 9B shows the responses in a similar cell loaded with Fura-2 and BAPTA. These results clearly demonstrate that the presence of suYcient BAPTA practically eliminates the rapid and transient rises in [Ca2þ]i elicited by an agonist.
5. Measuring [Ca2þ] with Fluorescent Indicators
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Fig. 9 BuVering action of intracellular BAPTA on changes in [Ca2þ]i. (A) Changes in [Ca2þ]i of a
REF52 cell treated with 1 mM bradykinin and then 1 mM Br-A23187. (B) Changes in [Ca2þ]i of a REF52 cell, preloaded with BAPTA, in response to the same treatments as in (A). Cells were loaded with 1 mM Fura-2 AM in Pluronic dispersion for 85 min at 25 C. For (B), 20 mM BAPTA AM was also present in the incubation medium. Experiments were done in HBSS.
Indeed, even the massive rise resulting from a combination of Ca2þ influx and discharging of intracellular calcium stores mediated by Br-A23187 is suppressed substantially by the buVering action of BAPTA.
2. Possible Controls for the Use of BAPTA Similar to EGTA, BAPTA is a chelator not only for Ca2þ but also for other multivalent metal cations. Thus, one may wish to ensure that any inhibitory eVect observed when using BAPTA is caused strictly by the ability of BAPTA to buVer Ca2þ, and not because it is scavenging other biochemically important metal ions such as Zn2þ. The reagent used to control for heavy metal scavenging by BAPTA is TPEN (N,N,N0 ,N0 -tetrakis(2-pyridylmethyl)ethylenediamine) (Fig. 10), a membrane-permeant metal ion chelator that shows a marked preference for binding heavy metal cations over Ca2þ (Anderegg et al., 1977). Whereas the Kd(Ca2þ) of TPEN is 40 mM (Arslan et al., 1985), Kd(Zn2þ) is 2.6 10 16 M (Anderegg and Wenk, 1967). This enormous selectivity for binding heavy metal ions over Ca2þ enables TPEN to scavenge heavy metal ions very eVectively, even in the presence of
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N N N
N
CO2− −O2C N
−O2C CO2− N
−O2C CO2− N
N N
O
O
O H3C
TPEN
BAPTA
Half-BAPTA
Fig. 10 Structures of TPEN, BAPTA, and half-BAPTA.
millimolar levels of Ca2þ. That TPEN is membrane-permeant means it can be applied without using any special procedures. Dry DMSO can be used to prepare stock solutions of TPEN. Typically, TPEN is used in aqueous medium at a concentration of 10 6–10 5 M. BAPTA loading through the AM ester is very eYcient; high concentrations of BAPTA may be accumulated intracellularly. Thus, ascertaining that observed inhibitory eVects are not the result of cytotoxicity arising from the presence of high concentrations of a foreign organic anion may be important. In this case, the control reagent is N-(o-methoxyphenyl)iminodiacetic acid,19 sometimes referred to as ‘‘half-BAPTA.’’ As can be seen from Fig. 10, half-BAPTA is essentially chemically identical to BAPTA except that the molecule is only half of BAPTA. Because the full tetracarboxylate structure of BAPTA is crucial for Ca2þ binding, half-BAPTA, lacking such a structure, shows only very weak aYnity for Ca2þ (Kd 3 mM; J.P.Y. Kao, unpublished results). Half-BAPTA is thus expected to mimic BAPTA in all chemical respects except for the ability to buVer Ca2þ at physiological concentrations. The AM ester of half-BAPTA is sporadically available from commercial vendors. Cell loading via the AM ester can be done as described previously for other AM esters.20
V. Conversion of Indicator Fluorescence Signal into Values of [Ca2þ] Although raw fluorescence signals from intracellularly trapped Ca2þ indicators can be informative in a qualitative way, one still must perform some calibration before even semiquantitative estimates of [Ca2þ]i can be made. Basic principles of,
19
A trivial name is anisidine-N,N-diacetic acid. Because it is processed by esterases to generate only the Ca2þ-insensitive half-BAPTA and yet it is processed intracellular in the same way that all AM esters are, half-BAPTA AM can also be used as a control for possible artifacts from AM ester hydrolysis. 20
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as well as experimental procedures for, calibration for ratiometric and nonratiometric indicators are discussed in this section.
A. Calibrating a Nonratiometric Fluorescent Indicator For a nonratiometric indicator that increases fluorescence emission on binding Ca2þ, the free Ca2þ concentration is given by 2þ F Fmin ð1Þ Ca ¼ Kd Fmax F where Fmin is the indicator fluorescence intensity at zero [Ca2þ] (when all indicator molecules in the sample are Ca2þ-free), Fmax is the indicator fluorescence at saturatingly high [Ca2þ] (when all indicator molecules are present as the Ca2þ-bound form), and F is the measured fluorescence intensity for which we wish to find a corresponding value of [Ca2þ]. To arrive at a correspondence between measured F and [Ca2þ]i, Kd, Fmin, and Fmax all must be known. Whereas Kd usually is predetermined in vitro, Fmin and Fmax must be obtained in situ. The most straightforward approach would be to try to equilibrate the indicator-loaded cell with solutions that contain ‘‘zero’’ [Ca2þ] and then high [Ca2þ]. In practice, however, deficiencies inherent in a nonratiometric indicator make this approach unattractive. Interpretation of intensity changes is confounded by dye leakage, which causes the total indicator fluorescence from the cell (and therefore Fmin and Fmax) to decrease with time. This basic flaw of nonratiometric indicators means that obtaining good quantitative estimates of [Ca2þ]i in cells in which dye leakage or extrusion occurs at significant rates would be diYcult. In such cases, a laborious calibration would not be justified. An alternative semiquantitative calibration procedure developed for Fluo-3 is discussed next. The calibration procedure for Fluo-3 depends on the fact that, in vitro, FMn ¼ 0.2Fmax, where FMn is the fluorescence intensity when Fluo-3 is saturated completely with Mn2þ (Kao et al., 1989; Minta et al., 1989). That Fmax ¼ 100Fmin is also known from in vitro measurements. Because both Fmax and Fmin can be expressed in terms of FMn, the only parameter that must be determined experimentally is FMn. In situ calibration then consists of: 1. applying micromolar levels of ionomycin or Br-A23187 to increase permeability of the cell to divalent metal ions; 2. adding suYcient MnCl2 (typically twice the concentration of Ca2þ in solution)21 to ensure saturation of intracellular Fluo-3; and 21 In Step 2, it is best if the medium contains no carbonate, bicarbonate, or phosphates, which can form insoluble precipitates with Mn2þ and thus reduce the concentration of free Mn2þ. Moreover, the light scattering by the particulate precipitates can add considerable noise to the fluorescence signal.
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3. permeabilizing the cell with digitonin to release Fluo-3 to permit estimation of fluorescence background. Because the fluorescence intensity measured after Step 3 is just the background signal (including cellular autofluorescence), whereas the intensity after Step 2 is (FMn þ background), one can obtain FMn by subtraction. The calibration procedure described here is based on the following assumptions: (1) indicator fluorescence intensity is not diminishing rapidly as a result of leakage; (2) the fluorescence properties (Fmin, Fmax, and FMn) of the indicator are known from in vitro measurements and are the same in cells as in vitro; and (3) the Kd of the indicator is also the same in cells as in vitro. Quin2 is an example of an indicator the fluorescence of which is quenched completely by heavy metal ions. For calibration of such an indicator, see the review on Quin2 by Tsien and Pozzan (1989).
B. Calibrating a Ratiometric Fluorescent Indicator A dual-wavelength ratiometric indicator allows excitation spectral intensity or emission spectral intensity of the indicator to be monitored at two diVerent wavelengths. If F1 is the fluorescence intensity at wavelength l1, F2 is the fluorescence intensity at wavelength l2, and R ¼ F1/F2, then the free Ca2þ concentration can be shown to be (Grynkiewicz et al., 1985) R Rmin sf;2 ð2Þ ½Ca2 þ i ¼ Kd Rmax R sb;2 where Rmin is the limiting value of the ratio R when all the indicator is in the Ca2þfree form and Rmax is the limiting value of R when the indicator is saturated with Ca2þ.22 Experimentally, the factor sf,2/sb,2 is simply the ratio of the measured fluorescence intensity when all the indicator is Ca2þ-free to the intensity measured when all the indicator is Ca2þ-bound, with both intensity measurements taken at l2. On the right side of Eq. (2), with the exception of Kd, which is an intrinsic property of the indicator, all other terms are ratios of intensities; in forming these ratios, problems associated with cell shape and dye concentration changes cancel. Using Eq. (2) to calculate [Ca2þ] requires that Kd be known and that Rmin, Rmax, and sf,2/sb,2 be determined experimentally. A typical calibration entails 1. increasing Ca2þ permeability of the cell with ionomycin or Br-A23187 in the presence of ‘‘zero’’ extracellular Ca2þ (EGTA or BAPTA in nominally Ca2þfree medium; Section IV.B.2), so all intracellular Ca2þ could be depleted; 22 Usually, l1 and l2 are chosen so that intensity measured at l1 consists mostly of fluorescence emitted by the Ca2þ-bound form of the indicator, whereas intensity at l2 consists mostly of fluorescence from the Ca2þ-free form. Choosing the wavelength pair in this way increases the diVerence between Rmin and Rmax, making it possible to map [Ca2þ] onto a wider range of R values.
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2. increasing extracellular [Ca2þ] in the presence of Ca2þ ionophore so that Ca2þ could enter the cell to saturate intracellular indicator; 3. permeabilizing the cell with digitonin (at concentrations prescribed in Section III.B.2) to release cytosolic dye so the background signal may be measured.23 Although the procedure seems straightforward, a few empirical findings are helpful in performing a successful calibration: 1. Many cell types do not tolerate severe calcium deprivation well. During Step 1, these cells often become fragile or leaky or, in the case of adherent cells, detach from the substrate. In many cases, however, one can compensate for the total absence of Ca2þ by supplementation with elevated concentrations of Mg2þ. Thus, raising the extracellular [Mg2þ] to 5–20 mM can help maintain cell integrity during a long calibration. Although Mg2þ should bind Fura-2 to a limited extent and slightly alter its fluorescence spectrum (Grynkiewicz et al., 1985), in practice Rmin is not aVected significantly by Mg2þ supplementation. 2. Because ionomycin and Br-A23187 become very ineYcient at Ca2þ transport when intra- and extracellular free Ca2þ concentrations are below micromolar levels, depleting the cell of Ca2þ entirely is quite diYcult. Therefore, one often must wait a long time for true Rmin to be reached in Step 1. In the example shown in Fig. 11, the interval between ionomycin addition and attainment of Rmin was in excess of 90 min. 3. Step 2 could be performed in two ways. One could add, in combination with fresh ionophore if desired, suYcient Ca2þ to bind to all the EGTA or BAPTA that was introduced in Step 1 and still have a large excess of free extracellular Ca2þ, as well as suYcient TRIS base to counteract any acidification arising from the Ca2þEGTA binding reaction. Alternatively, one could replace the medium from Step 1 with nominally Ca2þ-free medium and then add a large excess of Ca2þ in combination with a fresh dose of ionophore. The aim is to initiate massive Ca2þ influx into the cell at a rate that overcomes any Ca2þ extrusion mechanism that the cell may mobilize. In practice, concentrations of ionophore ranging from 10 6 to 10 5 M and external [Ca2þ] in the range of one to several tens of millimolar can be used. 23 At controlled concentrations, digitonin releases primarily cytosolic dye whereas compartmentalized dye remains with the permeabilized cell and would be subtracted out as background. The assumption is that intraorganellar [Ca2þ] is significantly higher (> mM) than cytosolic [Ca2þ], so compartmentalized indicator would be essentially completely Ca2þ-bound and thus contribute a constant background to the measured 340- and 380-nm fluorescence signals from the cell. This assumption would fail if significant amounts of dye are compartmentalized into organelles that do not have high luminal [Ca2þ]. An alternative approach to obtaining a background reading is to add MnCl2 (at a concentration equal to or greater than the Ca2þ concentration in the medium) at the same time as the digitonin so that compartmentalized dye can also be quenched as ionophores transport Mn2þ into the organelles. Using such an approach assumes that cellular autofluorescence is the true background and ignores the contribution of compartmentalized dye to the background.
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4. A stable baseline is obtained quickly only if the dye released from cells by digitonin permeabilization is swept rapidly away from the region directly above the microscope objective. Otherwise, fluorescence from the released dye still will be captured by the objective and, thus, contribute to the measured background. Once swept away and diluted into the bulk medium, the released dye contributes negligibly to the background.
A
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Fig. 11 Procedure for in situ calibration of intracellular Fura-2. (A) Fluorescence intensity traces acquired at 340- and 380-nm excitation. Time marker arrow correspond to (1) addition of 50 nM vasopressin; (2) exchange into Ca2þ-free phosphate-buVered saline (PBS) containing 10 mM MgCl2, 2 mM EGTA, pH 7.4; (3) addition of 10 mM ionomycin; (4) exchange into nominally Ca2þ-free saline, pH 7.4; (5) addition of 10 mM ionomycin þ 20 mM CaCl2; and (6) 20 mM digitonin. Dotted lines mark fluorescence levels corresponding to various parameters discussed in Section V.B. (a.u. = arbitrary units). (B) F340/F380 ratio trace derived from the data in (A). Dotted lines mark Rmin and Rmax (0.566 and 16.6, respectively, in this experiment). The parameter sf,2/sb,2 is 10.7. Inset. The portion of the trace from 20 to 118 min at higher resolution on the vertical scale to reveal the gradualness with which Rmin is approached. (C) [Ca2þ]i trace derived from the ratio trace by using Eq. (2) in Section V.B. Only the first 40 min of the experiment are shown. This REF52 cell was incubated with 1 mM Fura-2 AM in Pluronic dispersion in HBSS for 90 min at 25 C before being transferred to fresh HBSS for measurement.
Elevation of [Ca2þ]i by ionophore can lead to rapid cell lysis and loss of indicator, sometimes before Rmax can be determined confidently. Almost paradoxically, raising the extracellular [Ca2þ] to 10–30 mM (rather than just a few mM) in this procedure appears, in some cases, to have a protective eVect on cell structure so lysis is deferred and Rmax can be reached. If high extracellular [Ca2þ] is used, the medium should be free of phosphate salts, bicarbonate/carbonate, and even sulfate, since these ions can form precipitates with Ca2þ. Typical data from an experiment performed on a REF52 cell loaded with Fura2 are shown in Fig. 11. Shown in Fig. 11A are the two raw data traces, F 0 340 and F 0 380, collected when the cell is excited alternately with 340-nm and 380-nm light. The fluorescence signals measured after digitonin permeabilization are the background intensities, BG340 and BG380, that must be subtracted from the respective traces to yield the true F340 and F380 (i.e., F340 ¼ F 0 340 BG340 and F380 ¼ F 0 380 BG380). The ratio trace is simply a point-by-point division,
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R ¼ F340/F380 and is shown in Fig. 11B. Rmin is the limiting value of R that is reached during Ca2þ deprivation, whereas Rmax is the limiting value of R reached after treatment with ionophore at high [Ca2þ].24 The factor sf,2/sb,2 is essentially (F 0 f,380 BG380)/(F 0 b,380 BG380). Using these experimentally derived parameters and a predetermined Kd (224 nM; Grynkiewicz et al., 1985) in Eq. (2), one can convert the F340/F380 ratio trace into a plot of [Ca2þ]i as a function of time (Fig. 11C). This procedure has the advantage that all spectroscopically derived parameters, namely Rmin, Rmax, and sf,2/sb,2, that are especially sensitive to environmental changes are determined in situ with the indicator residing in the intracellular environment. Only the equilibrium dissociation constant is determined in vitro. Rmin determined by Ca2þ deprivation is assumed to be the true value. In view of the ineVectiveness of currently available ionophores at low [Ca2þ], one would be justified in concluding that true Rmin would be diYcult to reach25 and that Rmin is easy to overestimate. An overestimate of Rmin results in underestimation of [Ca2þ]. Finally, it is worthwhile to examine the eVects of errors in Rmin, Rmax, and sf,2/sb,2 on the derived value of [Ca2þ]. For simplicity, one assumes that errors in the three parameters are independent. Because sf,2/sb,2 is related linearly to [Ca2þ] (see Eq. (2)), a percentage error in sf,2/sb,2 translates into the same percentage error in [Ca2þ]. Inspection of Eq. (2) reveals that errors in Rmin should aVect primarily low values of [Ca2þ] (corresponding to R values near Rmin). Error in Rmax, on the other hand, aVects the way in which all the R values are scaled and, therefore, should influence all derived values of [Ca2þ]. These expectations are borne out by calculation.26
24 From Fig. 11B, the ratio values near Rmax are seen to oscillate significantly because, at saturating [Ca2þ], the fluorescence of the indicator excited at 380 nm (Fb,380 ¼F 0 b,380 BG380) is very weak and cannot be determined with high precision. In forming the ratio, because Fb,380 is a small number and occurs in the denominator, noise fluctuations in Fb,380 become magnified into large-amplitude fluctuations in Rmax. Therefore, one must average a large number of points to obtain a reliable estimate of Rmax. Alternatively, the fluorescence intensity data (both F340 and F380) can be smoothed first before a ratio is formed. 25 Rather than estimating Rmin directly from the lowest values attained in the ratio trace, curvefitting the portion of the ratio trace that represents the slow descent towards Rmin is also a reasonable approach. As expected, Rmin obtained by exponential curve-fitting is somewhat lower than that estimated directly from the ratio trace. 26 When one uses parameters similar to those for Fura-2 in REF52 cells as determined on our instrument (Rmin ¼ 0.5, Rmax ¼ 15, and sf,2/sb,2 ¼ 12), a 10% overestimation of Rmin leads to 19% underestimation of [Ca2þ] at 50 nM, 10% at 100 nM, and 2% at 500 nM. A 10% overestimation of Rmax leads to underestimation of [Ca2þ] by 9.5% at 50 nM, 10.9% at 500 nM, and 12.5% at 1 mM. A 10% underestimation of Rmax results in overestimation of [Ca2þ] by 11.8% at 50 nM, 14% at 500 nM, and 16.5% at 1 mM.
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2.5
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Fig. 12 Two conventions, F/F0 and DF/F0, for reporting fluorescence changes relative to baseline fluorescence intensity. Note: DF/F0 ¼ F/F0 1.
VI. Reporting Indicator Fluorescence Intensity Changes without Calibration A. Reporting Relative Changes in Fluorescence: F/F0 and DF/F0 With the widespread use of nonratiometric indicators, which are diYcult to calibrate, it has become common to report not [Ca2þ], but rather indicator fluorescence changes. The convention is to report either the fluorescence intensity relative to baseline intensity (F/F0), or the change in fluorescence intensity relative to baseline intensity (DF/F0 ¼ (F F0)/F0). Figure 12 illustrates these two conventions. From the above definitions and from the graphs in Fig. 12, it is apparent that the two reporting conventions are simply related: DF/F0 ¼ F/F0 1. It is important to stress that in order for these relative measurements to be meaningful, F and F0 should be intensities that have been background-subtracted.
B. Caveat in Interpreting Relative Fluorescence Changes: Indicator Fluorescence is Not a Linear Function of [Ca2þ] Because a nonratiometric indicator becomes brighter when it binds Ca2þ, an increase in indicator fluorescence implies an increase in [Ca2þ]. Once fluorescence intensity data have been converted into relative changes, however, there is perhaps
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a natural tendency to regard the relative change in intensity as reflecting an equivalent relative change in [Ca2þ]. For example, a doubling of intensity relative to baseline (F/F0 ¼ 2 or DF/F0 ¼ 1) is often used to infer a doubling of [Ca2þ]. Such an inference should never be made because it is always incorrect. A quantitative analysis is presented below. As shown in Appendix 2, the total fluorescence, FT, emitted by a solution of Ca2þ indicator is governed by the expression FT / QCaIn eCaIn fCaIn þ QIn eIn ð1 fCaIn Þ
ð3Þ
where QCaIn and QIn are the fluorescence quantum eYciencies of the Ca -bound and Ca2þ-free forms of the indicator, respectively, eCaIn and eIn are the extinction coeYcients of the two forms of the indicator at the excitation wavelength, and fCaIn is the fraction of the indicator that is in the Ca2þ-bound form. Knowing that fCaIn ¼ [Ca2þ]/([Ca2þ] þ Kd), we can rewrite the expression to show its dependence on [Ca2þ] more explicitly: 2þ Ca FT / QIn eIn þ ðQCaIn eCaIn QIn eIn Þ 2þ ð4Þ Ca þ Kd 2þ
The only variable in the expression is [Ca2þ]; all other parameters, being intrinsic characteristics of a particular indicator, are constants. The above expression shows that whereas [Ca2þ] can range from 0 to any arbitrary positive value, the total fluorescence, FT is bounded. This behavior is shown in Fig. 13. When [Ca2þ] ¼ 0, all of the indicator is Ca2þ-free, and the fluorescence has a minimum value that depends on the intrinsic brightness (QIneIn) of the Ca2þ-free form of the indicator. At saturating [Ca2þ] ([Ca2þ] Kd), all of the indicator is Ca2þ-bound, and the fluorescence has a maximum value that depends on the intrinsic brightness (QCaIneCaIn) of the Ca2þ-bound form of the indicator. Once the indicator molecules are saturated, further increasing [Ca2þ] brings no increase in fluorescence. Therefore, as can be seen from Eq. (4) and Fig. 13, fluorescence intensity is a nonlinear function of [Ca2þ]. This nonlinearity is the reason that a relative change in indicator fluorescence does not imply an equal relative change in [Ca2þ]. Figure 13 shows that the discrepancy depends on the extent to which the indicator is already bound to Ca2þ: Starting from a relatively low [Ca2þ], increasing [Ca2þ] by an increment, DCa1, results in a fluorescence increase, DF1. From the now-higher [Ca2þ], a further identical increment of DCa2 (¼ DCa1) brings a much smaller fluorescence increase, DF2. The error in using relative fluorescence changes to infer relative [Ca2þ] changes can be analyzed quantitatively for a specific example. Fluo-4 is a nonratiometric indicator that is commonly used with 488-nm excitation. The extinction coeYcient of Fluo-4 changes only by a few percent upon binding Ca2þ (eIn eCaIn ¼ 77,000 M 1 cm 1 at 488 nm); the Ca2þ-bound form is at least 100 times more fluorescent than the Ca2þ-free form (QCaIn ¼ 0.14, QIn 0.0014); and Kd ¼ 345 nM. The quantitative relationship between [Ca2þ] and fluorescence can
5. Measuring [Ca2þ] with Fluorescent Indicators
141 Upper bound ∝ QCaIn εCaIn
ΔF2
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ΔF1
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ΔCa1
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Fig. 13 Indicator fluorescence intensity is a nonlinear function of [Ca2þ]. At [Ca2þ] ¼ 0, all indica-
tor molecules in solution are in the Ca2þ-free form, and indicator fluorescence is at the lower bound. Whereas [Ca2þ] can range from 0 to any arbitrarily large value, indicator fluorescence cannot exceed an upper bound, which is reached when all indicator molecules in solution are in the Ca2þ-bound form. The relationship between fluorescence intensity and [Ca2þ] is hyperbolic. The consequence is that successive equal increments in [Ca2þ] do not result in equal increments of fluorescence intensity (compare the fluorescence increments DF1 and DF2 resulting from two equal increments in [Ca2þ]).
be calculated by using these parameters in Eq. (4). Figure 14 shows the relative change in Fluo-4 fluorescence for diVerent increments in [Ca2þ], up to a 10-fold change ([Ca2þ]/[Ca2þ]0 ¼ 10). Because resting [Ca2þ]i is typically in the range 50– 100 nM, the starting [Ca2þ] was assumed to be [Ca2þ]0 ¼ 75 nM for the calculation. Figure 14 shows clearly that the relative change in fluorescence is never a good measure of the true relative change in [Ca2þ]. F/F0 significantly underestimates [Ca2þ]/[Ca2þ]0, and the error increases severely as the change in [Ca2þ] becomes larger.
VII. Measuring [Ca2þ] in Mitochondria As mentioned in Section II, when cells are incubated with the AM ester of Rhod-2, the indicator preferentially loads into mitochondria. The structures of two fluorescent dyes, TMRM and TMRE, which also accumulate preferentially into mitochondria, and Rhod-2 AM are shown in Fig. 15A. The positively charged structure in these molecules that enables preferential loading into mitochondria is highlighted with thick lines in Fig. 15A. Figure 15B shows that, rather than
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Fig. 14 Specific example illustrating that a relative change in fluorescence (F/F0) of the indicator, Fluo-4, does not accurately reflect the true relative change in [Ca2þ] ([Ca2þ]/[Ca2þ]0).
A
B
Fig. 15 (A) Structures of Rhod-2 AM (bromide salt), as well as TMRM and TMRE (percholorate salts), two dyes that accumulate preferentially into mitochondria. Note that in each case, the dye molecule bears a permanent positive charge. (B) A series of related resonance structures showing that the positive charge can be located on diVerent atoms in the molecule; that is, the charge is delocalized.
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being isolated on a single atom, the positive charge can reside on many diVerent atoms in the structure—that is, the positive charge is delocalized over the entire highlighted structure. A hydrophobic organic ion whose charge is delocalized can pass through lipid membranes. Mitochondria maintain a remarkably negative membrane potential—the mitochondrial lumen is typically at 150 to 200 mV relative to the cytosol. Therefore, Rhod-2 AM, which is a hydrophobic organic cation whose positive charge is delocalized, can permeate through the plasma membrane and the mitochondrial membranes and preferentially partition into the negative lumen of mitochondria. In mitochondria, cleavage of AM ester groups by esterases liberates the Ca2þ-sensitive form of Rhod-2 (bearing multiple nondelocalized negative charges), which is not membrane-permeant and thus trapped in the mitochondrial lumen. Therefore, Rhod-2 can be used to monitor intramitochondrial Ca2þ signals (Babcock et al., 1997; Tsien and Bacskai, 1995).
A. Estimating the Fraction of Intracellular Rhod-2 Indicator that Resides in Mitochondria While Rhod-2 can be preferentially loaded into mitochondria, the discrimination against loading into other subcellular compartments is imperfect. Some intracellular Rhod-2 is expected to reside in the cytosol and nonmitochondrial organelles. A simple procedure based on diVerential permeabilization of cellular membranes can be used to estimate the fraction of intracellular Rhod-2 that actually resides in mitochondria. The procedure is a modification of the one described in Section III.B.2. The procedure consists of monitoring total Rhod2 fluorescence from a cell or a group of cells bathed in low-Ca2þ medium and treating the cells sequentially with (1) a Ca2þ ionophore (ionomycin or BrA23187), (2) the mild detergent digitonin to permeabilize the plasma membrane, and (3) a strong detergent, for example, Triton X-100 or sodium dodecyl sulfate (SDS), to permeabilize all membranes. Figure 16 shows the procedure being applied to a vagal sensory neuron that had been incubated with 1 mM Rhod2 AM for 1 h at room temperature. Application of ionomycin abolishes significant diVerences in [Ca2þ] between diVerent subcellular compartments. This ensures that Rhod-2 in all compartments is at comparable levels of Ca2þ-binding, and thus would contribute fluorescence intensity in proportion to their actual content in each compartment. Once the fluorescence reaches a steady baseline after ionomycin treatment, digitonin permeabilization of the plasma membrane allows cytosolic Rhod-2 to escape,27 giving a decrement in total fluorescence (labeled ‘‘C’’ in Fig. 16). Subsequent permeabilization of all cellular membranes by SDS allows 27 Digitonin treatment leads to release of Rhod-2 from the nucleus as well. The nuclear pores have a size exclusion limit of 35–40 kDa; molecules with molecular mass less than the exclusion limit can freely exchange between the nucleoplasm and cytosol. Therefore, with respect to low-molecular-mass solutes such as simple ions (e.g., Ca2þ, Naþ, Cl) and small organic molecules (e.g., glucose, ATP, fluorescent indicators), the nucleo-cytoplasm functions as a single ‘‘cytosolic’’ compartment.
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Rhod-2 fluorescence (arbitrary units)
144
60 s
C
M + NM Iono Digit SDS 0-Ca/BAPTA
Fig. 16 A diVerential permeabilization experiment for estimating subcellular fractions of Rhod-2
indicator. A rabbit vagal sensory neuron was incubated with 1 mM Rhod-2 AM for 1 h at 23 C and then bathed in nominally Ca2þ-free physiological saline to which 2 mM Na4BAPTA was added (0-Ca/ BAPTA). The total Rhod-2 fluorescence from the cell was monitored. Ionomycin (Iono, 2 mM) was applied to dissipate Ca2þ gradients between subcellular compartments. Digitonin (Digit, 20 mM) permeabilized the plasma membrane selectively to release cytosolic Rhod-2 (intensity decrement marked ‘‘C’’). Sodium dodecyl sulfate (SDS; 0.25%, w/v) permeabilized all cellular membranes to release Rhod-2 from organellar compartments (decrement marked ‘‘M þ NM’’). The durations of reagent applications are indicated by the bars at the bottom.
Rhod-2 to escape from mitochondria as well as nonmitochondrial organelles; this causes a further decrement in fluorescence (labeled ‘‘M þ NM’’ in Fig. 16). For four neurons tested, the ratio of the noncytosolic fraction, M þ NM, to the cytosolic fraction, C, was M þ NM ¼ 5:36 C This provides one required algebraic condition; a second condition is M þ NM þ C ¼ 1 that is, intracellular Rhod-2 must be in the mitochondria, in nonmitochondrial organelles, or in the cytosol. Since there are three variables, a third algebraic condition is required, and this can be obtained by performing the permeabilization experiment on cells whose incubation with Rhod-2 AM had been done in the presence of a protonophore (e.g., CCCP, FCCP, 2,4-dinitrophenol),28 which 28 It is advisable to use oligomycin (e.g., 10 mM), a blocker of the mitochondrial F1F0-ATP synthase, in conjunction with the protonophore. In discharging the mitochondrial membrane potential, the protonophore eliminates the Hþ electrochemical gradient that is used by the ATP synthase to generate ATP. This causes the ATP synthase to run in reverse—as an ATPase—and rapidly deplete cellular ATP. Oligomycin, by blocking ATPase action, helps to preserve the cellular ATP pool.
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abolishes the mitochondrial membrane potential and thus eliminates the driving force for preferential partition of Rhod-2 AM into mitochondria. In these cells with depolarized mitochondria, the fluorescence decrement caused by digitonin still represents loss of cytosolic Rhod-2 (C), but the decrement caused by SDS may be attributed to Rhod-2 loss from nonmitochondrial organelles (NM). In four neurons loaded in the presence of 5 mM CCCP, NM ¼ 0:623 C which provides the last algebraic condition required to solve for the three unknowns, C, NM, and M. Using all the three conditions together yields C ¼ 0.156, NM ¼ 0.097, and M ¼ 0.747—about 75% of intracellular Rhod-2 reside in mitochondria, with 15% in the cytosol and 10% in other organelles.29
B. Minimizing Rhod-2 Loading in the Cytosol Having a few percent of Rhod-2 residing in nonmitochondrial organelles is likely to be unimportant. The luminal [Ca2þ] in these organelles (e.g., lysosomes, ER, etc.) is not expected to change markedly. Therefore, the Rhod-2 fluorescence signal from these organelles should not change significantly during an experiment and thus can be considered operationally to be part of the background fluorescence. In contrast, Rhod-2 in the cytosol, although much less than that in the mitochondria, may contribute a contaminating cytosolic Ca2þ signal when one attempts to measure mitochondrial [Ca2þ]. The cytosolic fraction can be minimized by taking advantage of the ubiquitous cellular transporters that extrude organic anions from the cytosol into the extracellular space (this is the process discussed in Section III.C). After they have been incubated with Rhod-2 AM at room temperature, cells can be transferred into medium containing no AM ester and incubated for 20–30 min at 37 C to accelerate extrusion of Rhod-2 from the cytosol. Thereafter, the Ca2þ-responsive Rhod-2 signal should be predominantly mitochondrial. 29 This likely underestimates mitochondrial loading, because we assumed that in the presence of protonophore, Rhod-2 loading into mitochondria was negligible. Without the benefit of the mitochondrial membrane potential, Rhod-2 AM can still diVuse into the depolarized mitochondria and be processed by esterases therein. Therefore, the decrement caused by SDS should contain both contributions from nonmitochondrial organelles and from passively loaded, depolarized mitochondria. If we assume that on a per-volume basis, the cytosol and mitochondria have comparable capacity to process AM esters, then the Rhod-2 content in depolarized mitochondria relative to that in the cytosol should reflect the ratio of the mitochondrial and cytosolic volumes. Depending on cell type, mitochondria-to-cytosol volume ratio can range from 1/5 to 1/3. Using the lower figure changes the third algebraic condition to (C/5 þ NM)/C ¼ 0.623, leading to the modified estimates: C ¼ 0.156, NM ¼ 0.066, and M ¼ 0.778. Using the higher figure of 1/3 leads to the condition, (C/3 þ NM)/C ¼ 0.623, which yields C ¼ 0.156, NM ¼ 0.045, and M ¼ 0.798. Comparing these numbers with those obtained in the main text shows that this additional correction only changed the estimate by a few percent.
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ΔF/F0 = 0.5
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60 s
Caf 0-Ca
Mito Cyto
Fig. 17 Simultaneous measurement of Ca2þ signals in the cytosol and in mitochondria. A rabbit vagal sensory neuron was incubated with 1 mM each Rhod-2 AM and Fluo-3 AM for 1 h at 23 C and then superfused with nominally Ca2þ-free physiological saline (0-Ca). Rhod-2 and Fluo-3 fluorescence, excited at 543 and 488 nm, respectively, were imaged simultaneously by laser-scanning confocal microscopy. Data are represented as fluorescence change relative to baseline (DF/F0). A 5-s pulse of caVeine (Caf, 10 mM) was delivered by superfusion. The durations of reagent applications are indicated by the bars at the bottom.
C. Monitoring Cytosolic and Mitochondrial [Ca2þ] Simultaneously Because indicators whose AM esters are uncharged load primarily into the cytosol, while Rhod-2 preferentially loads into mitochondria, one can monitor Ca2þ signals in the two compartments simultaneously. For measuring cytosolic [Ca2þ], one should select an indicator whose excitation and emission wavelengths do not interfere with Rhod-2 measurement. Since Rhod-2 is a rhodamine-based indicator, a fluorescein-based indicator would be suitable for the cytosolic measurement (e.g., members of the Fluo family of indicators). Figure 17 shows an experiment where the cytosolic and mitochondrial Ca2þ signals are monitored simultaneously in a vagal sensory neuron being stimulated with a brief pulse of caVeine (an agonist that activates ryanodine receptor Ca2þ channels to release Ca2þ from Ca2þ stores in the ER). The cytosolic and mitochondrial Ca2þ transients have very diVerent decay kinetics: the time for the Ca2þ signal to decay by 80% was t80% ¼ 7.7 s in the cytosol and t80% ¼ 65.1 s in mitochondria.
VIII. Concluding Remarks Fluorescent Ca2þ indicators have contributed enormously to our understanding of intracellular calcium regulation. For those who are beginning to use these indicators, the technical details can seem bewildering. This compendium of
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common techniques has aimed to set in order the body of practical empirical knowledge that underlies successful measurements of [Ca2þ] through the use of fluorescent indicators.
Appendix 1. The fraction of a polybasic acid that exists in a particular state of protonation The native forms of the Ca2þ buVers and indicators discussed in this chapter are polybasic acids; that is, they are species with multiple dissociable protons (e.g., H4EGTA). Almost invariably, the fully deprotonated species is the one that actually binds Ca2þ with high aYnity; therefore it is useful to be able to estimate the fraction of the indicator or buVer in solution that actually exists in the fully deprotonated form. In general, deprotonation of a polybasic acid is characterized by a sequence of dissociation equilibria. For the specific example of a tetrabasic acid, H4A, the sequence of stepwise dissociation reactions and the corresponding equilibrium constants are: H4 A > Hþ þ H3 A H3 A > Hþ þ H2 A2 H2 A2 > Hþ þ HA3 HA3 > Hþ þ A4
½Hþ ½H3 A ½ H4 A þ ½H H2 A2 K2 ¼ ½H3 A ½Hþ HA3 K3 ¼ H2 A2 ½Hþ A4 K4 ¼ HA3 K1 ¼
If we define C0 to be the total concentration of the acid, irrespective of the state of protonation, then the fraction that is present as the fully deprotonated, tetraanionic form, A4, is 4 A ðA1:1Þ a4 ¼ C0 To derive a useful expression for a4, we first recognize that the total concentration, C0, encompasses the concentrations of all the possible protonated forms: C0 ¼ ½H4 A þ ½H3 A þ H2 A2 þ HA3 þ A4 ðA1:2Þ Because all the concentrations are related to each other through the dissociation equilibrium constants, K1 through K4, the concentration of any particular protonated species can be written in terms of the concentration of any other species. In the present case, it is convenient to express all the concentrations in terms of [A4]:
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½H 4 HA3 ¼ A K4 þ
ðA1:3Þ
½Hþ ½Hþ 2 4 HA3 ¼ A H2 A2 ¼ K3 K4 K3 ½ H3 A ¼
ðA1:4Þ
½Hþ ½Hþ 4 H2 A2 ¼ A K2 K3 K4 K2
ðA1:5Þ
4 ½Hþ ½Hþ ½H3 A ¼ A K1 K2 K3 K4 K1
ðA1:6Þ
3
4
½H4 A ¼
By using these four relations, the expression for the total concentration can be written in terms of [A4]: 4 ½Hþ ½Hþ 4 ½Hþ 4 ½Hþ 4 4 A A A A þ þ þ þ A K1 K2 K3 K4 K2 K3 K4 K3 K4 K4 ðA1:7Þ 4
C0 ¼
3
2
Dividing through by [A4] leads to C0 1 ½Hþ ½Hþ ½Hþ ½Hþ ¼ ¼ þ þ þ þ1 a4 K1 K2 K3 K4 K2 K3 K4 K3 K4 K4 A4 4
3
2
ðA1:8Þ
Writing the right side of Eq. (A1.8) as a fraction with a common denominator and then inverting the fraction gives the desired final expression a4 ¼
K1 K2 K3 K4 þ 4
þ 3
þ 2
½H þ ½H K1 þ ½H K1 K2 þ ½Hþ K1 K2 K3 þ K1 K2 K3 K4
:
ðA1:9Þ
An important feature of Eq. (A1.9) to notice is that each term in the expression actually represents the contribution of a particular protonated form, thus: K1 K2 K3 K4 , A4 ½Hþ K1 K2 K3 , HA3 ½Hþ K1 K2 , H2 A2 2
½Hþ K1 , H3 A 3
½Hþ , H4 A 4
This insight makes it easy to write the fraction of the polybasic acid that is in a particular form: the term representing the particular protonated form appears in the numerator, while the denominator is simply the sum of all the possible terms. For example, the fraction existing as HA3 is a3 ¼
½Hþ K1 K2 K3 ½Hþ þ ½Hþ K1 þ ½Hþ K1 K2 þ ½Hþ K1 K2 K3 þ K1 K2 K3 K4 4
3
2
and the fraction existing in the doubly deprotonated H2A2 form is
;
ðA1:10Þ
5. Measuring [Ca2þ] with Fluorescent Indicators
a2 ¼
149 þ 2
½H K1 K2 þ 4
þ 3
þ 2
½H þ ½H K1 þ ½H K1 K2 þ ½Hþ K1 K2 K3 þ K1 K2 K3 K4
ðA1:11Þ
The plots shown in Fig. 7 were generated using the above expressions for a2, a3, and a4 in conjunction with the four stepwise dissociation constants for EGTA. In footnote 14, it was stated that Kd represents the ‘‘absolute’’ or intrinsic dissociation constant characterizing the fully deprotonated form of the chelator (e.g., A4 in the case above). At any pH where not all of the chelator is in the fully deprotonated form, Kd must be corrected for the weakening eVect of acidic pH; the corrected, or ‘‘conditional,’’ dissociation constant is K0 d. As one would expect, the correction factor is the fraction of chelator that exists in the fully deprotonated form at the desired pH (e.g., a4 in the case of a tetrabasic acid like EGTA). Thus, for a tetrabasic chelator, 0
Kd ¼
Kd a4
ðA1:12Þ
The plots shown in Fig. 8 were generated using Eq. (A1.12).
Appendix 2. Deriving an expression for the amount of fluorescence emitted by a solution of fluorescent indicator Light absorption by a solution containing a light-absorbing molecule, such as a colorimetric or fluorescent indicator, is described by the Beer–Lambert Law: A ¼ log
I ¼ elc I0
ðA2:1Þ
where A is the absorbance (or ‘‘optical density’’) of the solution, I0 is the intensity of a light beam impinging on the solution, I is the intensity after the beam has passed through the solution (I0 I ¼ Iabs is the amount of light absorbed), e is the molar extinction coeYcient (also known as the molar absorptivity), l is the thickness of the solution through which the light beam passes, and c is the concentration of the lightabsorbing molecule. The equation can be rearranged to the exponential form: I ¼ I0 e2:303elc
ðA2:2Þ
By convention, e has units of M 1cm 1 (i.e., l mol 1cm 1), l is measured in cm, and c is measured in units of molarity (M, or mol l 1). For typical imaging experiments where fluorescent indicators are loaded into cells, numerical limits may be defined for the three parameters of interest: e < 50; 000 M1 cm1 l < 50 104 cm c < 100 106 M
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The basis for these limits is as follows. First, indicators are small molecules whose extinction coeYcients almost never surpass 50,000. Second, with extremely rare exceptions, cells do not exceed 50 mm in diameter. Third, incubation with AM esters usually achieves intracellular indicator concentration of several tens of micromolar, and when indicators are introduced through a whole-cell patch electrode, the concentration is usually kept below 100 mM to ensure that excessive Ca2þ buVering capacity is not introduced into the cell. Applying these limits gives 2:303elc < 0:0575 1 Knowing that for x 1, e x 1 x, we can recast Eq. (A2.2) as I ¼ I0 ð1 2:303elcÞ ¼ I0 2:303elcI0
ðA2:3Þ
which rearranges to a simple expression of the amount of light absorbed by the sample: I0 I ¼ Iabs ¼ 2:303elcI0
ðA2:4Þ
This approximate expression is accurate to within 1% for absorbances less than 0.06 (i.e., elc < 0.06). After absorbing a photon, a fluorescent molecule may reemit the absorbed quantum of energy as fluorescence. The probability that after absorbing a photon, a molecule will emit a photon of fluorescence is known as the quantum eYciency of fluorescence. Quantum eYciency is usually symbolized as Q or f (Greek phi). The amount of fluorescence emission should be the amount of light absorbed multiplied by the quantum eYciency: F ¼ 2:303QelcI0
ðA2:5Þ
In this expression, only Q and e are intrinsic molecular properties, and these can diVer substantially between the Ca2þ-free and Ca2þ-bound forms of the indicator (symbolized as In and CaIn, respectively). For typical nonratiometric indicators, QCaIn QIn, while eCaIn eIn. The total concentration of indicator, CT, is the sum of the concentrations of the Ca2þ-bound and Ca2þ-free forms: CT ¼ ½CaIn þ ½In
ðA2:6Þ
Making use of the dissociation equilibrium constant: 2þ Ca ½In 2þ CaIn > Ca þ In Kd ¼ ½CaIn we can deduce the fraction of indicator that is Ca2þ-bound, fCaIn, and the fraction that is Ca2þfree, fIn: 2þ Ca Kd and fIn ¼ 2þ ðA2:7Þ fCaIn ¼ 2þ Ca Ca þ Kd þ Kd
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and, by necessity, fCaIn þ fIn ¼ 1. Therefore, ½CaIn ¼ fCaIn CT and ½In ¼ fIn CT ¼ ð1 fCaIn ÞCT
ðA2:8Þ
The total fluorescence, FT, from a solution of indicator contains contributions from both CaIn and In forms: FT ¼ FCaIn þ FIn ¼ 2:303QCaIn eCaIn l½CaIn I0 þ 2:303QIn eIn l½In I0 ¼ 2:303QCaIn eCaIn lfCaIn CT I0 þ 2:303QIn eIn lfIn CT I0
ðA2:9Þ
¼ 2:303lCT I0 ½QCaIn eCaIn fCaIn þ QIn eIn fIn This shows that the total fluorescence depends on the intrinsic molecular properties of In and CaIn and the relative abundance of the two forms: FT / QCaIn eCaIn fCaIn þ QIn eIn fIn
ðA2:10Þ
Moreover, because the product, Qe, is a composite measure of a molecule’s ability to absorb light and then emit fluorescence, we may think of Qe as the ‘‘intrinsic brightness’’ of a fluorescent molecule. The brightness contribution of each indicator form to the total fluorescence is weighted by the relative abundance of each form. Finally, because fCaIn þ fIn ¼ 1, we can write FT / QCaIn eCaIn fCaIn þ QIn eIn ð1 fCaIn Þ
ðA2:11Þ
References Anderegg, G., and Wenk, F. (1967). Pyridinderivate als Komplexbildner. VIII. Die Herstellung je eines neuen vier- und sechsza¨hnigen Liganden. Helv. Chim. Acta 50, 2330–2332. Anderegg, G., Hubmann, E., Podder, N. G., and Wenk, F. (1977). Pyridinderivate als Komplexbildner. XI. Die Thermodynamik der Metallkomplexbildung mit Bis-, Tris- und Tetrakis[(2-pyridyl)methyl]aminen. Helv. Chim. Acta 601, 123–140. Arslan, P., Di Virgilio, F., Beltrame, M., Tsien, R. Y., and Pozzan, T. (1985). Cytosolic Ca2+ homeostasis in Ehrlich and Yoshida carcinomas. A new, membrane-permeant chelator of heavy metals reveals that these ascites tumor cell lines have normal cytosolic free Ca2+. J. Biol. Chem. 260, 2719–2727. Babcock, D. F., Herrington, J., Goodwin, P. C., Park, Y. B., and Hille, B. (1997). Mitochondrial participation in the intracellular Ca2+ network. J. Cell Biol. 136, 833–844. Di Virgilio, F., Steinberg, T. H., Swanson, J. A., and Silverstein, S. C. (1988). Fura-2 secretion and sequestration in macrophages. A blocker of organic anion transport reveals that these processes occur via a membrane transport system for organic anions. J. Immunol. 140, 915–920. Di Virgilio, F., Steinberg, T. H., and Silverstein, S. C. (1990). Inhibition of Fura-2 sequestration and secretion with organic anion transport blockers. Cell Calcium 11, 57–62. Grynkiewicz, G., Poenie, M., and Tsien, R. Y. (1985). A new generation of Ca2+ indicators with greatly improved fluorescence properties. J. Biol. Chem. 260, 3440–3450. Haugland, R. P. (1992). ‘‘Handbook of Fluorescent Probes and Research Chemicals.’’ Molecular Probes, Eugene, Oregon.
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Joseph P. Y. Kao et al. Hepler, P. K., and Callaham, D. A. (1987). Free calcium increases during anaphase in stamen hair cells of Tradescantia. J. Cell Biol. 105, 2137–2143. Kao, J. P. Y., Harootunian, A. T., and Tsien, R. Y. (1989). Photochemically generated cytosolic calcium pulses and their detection by fluo-3. J. Biol. Chem. 264, 8179–8184. Kao, J. P. Y., Alderton, J. M., Tsien, R. Y., and Steinhardt, R. A. (1990). Active involvement of Ca2+ in mitotic progression of Swiss 3T3 fibroblasts. J. Cell Biol. 111, 183–196. Liu, C.-M., and Hermann, T. E. (1978). Characterization of ionomycin as a calcium ionophore. J. Biol. Chem. 253, 5892–5894. Malgaroli, A., Milani, D., Meldolesi, J., and Pozzan, T. (1987). Fura-2 measurement of cytosolic free Ca2+ in monolayers and suspensions of various types of animal cells. J. Cell Biol. 105, 2145–2155. Martell, A. E., and Smith, R. M. (1974). ‘‘Critical Stability Constants,’’ Vol. I. Plenum, New York. McNeil, P. L. (1989). Incorporation of macromolecules into living cells. Meth. Cell Biol. 29, 153–173. McNeil, P. L. (2001). Direct introduction of molecules into cells. Curr. Protoc. Cell Biol. 20.1.1–20.1.7. Minta, A., Kao, J. P. Y., and Tsien, R. Y. (1989). Fluorescent indicators for cytosolic calcium based on rhodamine and fluorescein chromophores. J. Biol. Chem. 264, 8171–8178. Motoyama, K., Karl, I. E., Flye, M. W., Osborne, D. F., and Hotchkiss, R. S. (1999). Effect of Ca2+ agonists in the perfused liver: Determination via laser scanning confocal microscopy. Am. J. Physiol. 276, R575–R585. Niggli, E., Piston, D. W., Kirby, M. S., Cheng, H., Sandison, D. R., Webb, W. W., and Lederer, W. J. (1994). A confocal laser scanning microscope designed for indicators with ultraviolet excitation wavelengths. Am. J. Physiol. 266, C303–C310. Sako, Y., Sekihata, A., Yanagisawa, Y., Yamamoto, M., Shimada, Y., Ozaki, K., and Kusumi, A. (1997). Comparison of two-photon excitation laser scanning microscopy with UV-confocal laser scanning microscopy in three-dimensional calcium imaging using the fluorescence indicator Indo-1. J. Microsc. 185, 9–20. Tsien, R. Y. (1980). New calcium indicators and buffers with high selectivity against magnesium and protons: Design, synthesis, and properties of prototype structures. Biochemistry 19, 2396–2404. Tsien, R. Y., and Bacskai, B. J. (1995). Video rate confocal microscopy. In ‘‘Handbook of Biological Confocal Microscopy,’’ (J. B. Pawley, ed.), pp. 459–478. Plenum Press, New York. Tsien, R. Y., and Pozzan, T. (1989). Measurement of cytosolic free Ca2+ with quin2. Meth. Enzymol. 172, 230–262. Tsien, R. Y., Pozzan, T., and Rink, T. J. (1982). Calcium homeostasis in intact lymphocytes: Cytoplasmic free calcium monitored with a new, intracellularly trapped fluorescent indicator. J. Cell Biol. 94, 325–334.
CHAPTER 6
Genetically Encoded Probes for Measurement of Intracellular Calcium Michael Whitaker Institute of Cell and Molecular Biosciences Medical School, Newcastle University, Framlington Place Newcastle upon Tyne, United Kingdom
Abstract I. Introduction II. Genetically Encoded Sensors A. The Cameleon Family B. Camgaroos C. Pericam G-CaMP Family III. Applications of Genetically Encoded Sensors A. Targeting to Subcellular Locations B. Tissue-Specific Expression IV. Use of Genetically Encoded Calcium Sensors V. Conclusions References
Abstract Small, fluorescent, calcium-sensing molecules have been enormously useful in mapping intracellular calcium signals in time and space, as chapters in this volume attest. Despite their widespread adoption and utility, they suVer some disadvantages. Genetically encoded calcium sensors that can be expressed inside cells by transfection or transgenesis are desirable. The last 10 years have been marked by a rapid evolution in the laboratory of genetically encoded calcium sensors both figuratively and literally, resulting in 11 distinct configurations of fluorescent proteins and their attendant calcium sensor modules. Here, the design logic and performance of this abundant collection of sensors and their in vitro and in vivo use and performance are described. Genetically encoded calcium sensors have proved valuable in the measurement of calcium concentration in cellular METHODS IN CELL BIOLOGY, VOL. 99 Copyright 2010, Elsevier Inc. All rights reserved.
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0091-679X/10 $35.00 DOI: 10.1016/S0091-679X(10)99006-7
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Michael Whitaker
organelles, for the most part in single cells in vitro. Their success as quantitative calcium sensors in tissues in vitro and in vivo is qualified, but they have proved valuable in imaging the pattern of calcium signals within tissues in whole animals. Some branches of the calcium sensor evolutionary tree continue to evolve rapidly and the steady progress in optimizing sensor parameters leads to the certain hope that these drawbacks will eventually be overcome by further genetic engineering.
I. Introduction Small, fluorescent, calcium-sensing molecules have been enormously useful in mapping intracellular calcium signals in time and space, as chapters in this volume attest. Despite their widespread adoption and utility, they suVer some disadvantages. All low molecular mass fluorescent cytoplasmic calcium sensors are highly charged molecules, so cross the cell’s plasma membrane very poorly. They are placed into the cytoplasm by microinjection using fine-tipped micropipette or a patch clamp pipette in whole cell mode. This limits their utility. Cell-permeant fluorescent calcium sensors can be made by masking the charged carboxylic groups by forming acetoxymethyl (AM) esters. Once inside the cell, the ester bonds are cleaved, trapping the sensor in the cell. It is straightforward to bathe cells in culture with the aposensor at low concentration and these AM esters have been very widely used. One major drawback of the method is that the calcium sensor finds itself not only in the cytoplasm, but also in intracellular compartments such as the endoplasmic reticulum (ER) (Silver et al., 1992). Calcium concentrations are higher in the ER than in the cytoplasm, so this leads to a significant unwanted fluorescence signal from sensor in the ER that makes interpretation of the true cytoplasmic concentration changes diYcult. It is also very challenging to use lowmolecular-mass fluorescent calcium sensors in whole animals. For these reasons, genetically encoded calcium sensors that can be expressed inside cells by transfection or transgenesis are desirable. One such sensor is aequorin, a calcium-sensing protein found in the jellyfish Aequoria victoria. Originally, aequorin was isolated as a protein from jellyfish and placed inside cells by microinjection (Baker, 1978; Gilkey et al., 1978). More recently, a construct encoding recombinant aequorin has been used to express the aequorin apoprotein in cells directly (see Chapter 10). Aequorin is a luminescent molecule and at the concentrations used inside cells emits relatively few photons compared to fluorescent molecules at appropriate excitation intensities (Varadi and Rutter, 2002b). However, proteins that are fluorescent at the visible wavelengths best suited to fluorescence imaging are relatively rare. As it happens, A. victoria also expresses a fluorescent protein, green fluorescent protein (GFP), and it is the work that has produced the variously colored versions of GFP that has improved our knowledge of this fluorophore and led to recombinant fluorescent calcium sensors (Tsien, 2010).
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The first recombinant fluorescent calcium sensors were described by Tsien and Persecchini in 1997 (Miyawaki et al., 1997; Persechini et al., 1997; Romoser et al., 1997). They were based on a concatenation of a recombinant calcium-binding domain with GFP-derived fluorescent protein pairs. This approach has bred a family of these cameleon indicators, so called because they are based on a long tongue-like interaction between calmodulin (CaM) and a binding peptide and change color (Miyawaki et al., 1997). Later, when it was realized that the GFP beta-can structure lent itself to circular permutation without loss of function (Baird et al., 1999), insertion of a calcium-binding domain within the GFP (Baird et al., 1999) or concatenated to new N- or C-terminals (Nakai et al., 2001) led to a second family of calcium sensors based on the fluorescence of a single GFP-derived molecule, the camgaroos, pericams and their relatives. The last 10 years have been marked by a rapid evolution in the laboratory of these two families and their relatives, both figuratively and literally, as random mutagenesis and clonal selection in bacteria has on occasion been used to optimize the properties of the sensors (Griesbeck et al., 2001). This rapid diversification has generated not only continuing improvements in the performance of the sensors, but also a plethora of choice. Reviews have been written to track progress in the field (Barth, 2007; Demaurex and Frieden, 2003; Garaschuk et al., 2006; Griesbeck, 2004; Hires et al., 2008; KotlikoV, 2007; Mank and Griesbeck, 2008; Miyawaki, 2003a,b, 2005; Pozzan and Rudolf, 2009; Solovyova and Verkhratsky, 2002; Zacharias et al., 2000). Most of the new variants have first been tested by their makers in living cells as proof of principle rather than to answer substantial questions in biology. I shall first set out the evolution of this growing tribe of genetically encoded calcium sensing probes, dealing with the two broad families in turn and then describe their application and utility in various biological settings.
II. Genetically Encoded Sensors A. The Cameleon Family
1. Origins The family founders were described in three papers that followed rapidly in succession in 1997. Their conception was aided by previous work in which GFP had been altered by directed mutagenesis to produce diVerent colored variants with altered excitation and emission spectra (Heim et al., 1995). As an aside, these diVerently colored variants are sometimes referred to collectively as GFPs, though they are not green. Persechini’s group described a construct (FIP-CBsm) in which a red-shifted excitation variant of GFP (RSGFP; Delagrave et al., 1995, hereafter GFP) and blue fluorescent protein (BFP) are linked by a sequence that includes 17 amino acids from the calmodulin-binding domain of avian myosin light chain kinase (MLCK). This novel protein indirectly senses calcium concentrations inside
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cells, as when calcium increases, endogenous calmodulin becomes activated and binds to the MLCK calcium-binding domain. This in turn alters the disposition of the attached GFPs and leads to changes in Fo¨rster resonance energy transfer (FRET) between the blue and green proteins (Romoser et al., 1997). FRET is the phenomenon on which the cameleon sensor family relies. It occurs between closely apposed fluorophores that have overlapping emission and excitation spectra (Jares-Erijman Elizabeth and Jovin Thomas, 2003). In this example, the emission spectrum of BFP overlaps with the excitation spectrum of GFP. The extent of FRET depends on the degree of overlap between the two spectra, the orientation of the fluorescence dipoles and crucially, the distance between them. There is a very steep sixth power relationship with distance, so the energy transfer is very sensitive to distance between fluorophores over the range 1–10 nm (JaresErijman and Jovin, 2003). Calmodulin binds to the helical MLCK sequence by wrapping its two lobes around it (Ikura et al., 1992). In FIP-CBsm, the steric bulk of the calmodulin molecule when it binds to the MLCK peptide linker forces the BFP and RFP further apart and reduces FRET (Romoser et al., 1997). FRET can be measured in a variety of ways (Jares-Erijman Elizabeth and Jovin Thomas, 2003; Visser et al., 2010), but conceptually the simplest method is to excite the donor fluorophore, here BFP, and measure the emission of both the donor and the acceptor, here GFP. FRET takes place by nonradiative energy transfer, so high levels of FRET transfer energy from BFP to GFP, reducing BFP emission at around 440 nm and increasing GFP emission at 510 nm. Calmodulin binding reduces FRET, increasing emission at 440 nm and reducing emission at 510 nm. These changes can be expressed as a ratio of emission at the two wavelengths, a value independent of the concentration of the protein. In HEK-239 cells expressing FIP-CBsm, ratio changes (F510/F440) of around three- to fourfold could be observed after raising free intracellular calcium concentration with the calcium ionophore ionomycin (Romoser et al., 1997). FIP-CBsm relied on endogenous calmodulin to generate a calcium-sensitive FRET signal between GFPs. Tsien’s construct concatenated Xenopus laevis calmodulin and an MLCK calmodulin-binding peptide, M13 (Ikura et al., 1992), together between BFP and GFP and also in an analogous construct between two other GFP variants, enhanced cyan fluorescent protein (ECFP) and enhanced yellow fluorescent protein (EYFP). In this concatenated configuration, binding of calcium to calmodulin causes it to loop back toward the M13 peptide (the cameleon’s tongue) as it binds, reducing the distance between the two GFP variants and enhancing FRET (Miyawaki et al., 1997). This study beautifully exemplifies the power of the cameleon concept linked to selective mutagenesis: the original BFP/GFP construct (cameleon-1) worked well in vitro, but did not express suYciently in mammalian cells; the enhanced variant with mammalian codon usage (EBFP/EGFP—cameleon-2) showed much improved expression, but the best expression, brightness, and signal-to-noise data were seen with enhanced cyan and yellow variants of GFP (ECFP/EYFP—yellow cameleon-2). These benefits came, however, at the expense of a lower FRET change between calcium
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bound and unbound forms (1.5 vs. 1.8 when expressed as a ratio of emission wavelengths) and a greater pH sensitivity. Mutagenesis can also be applied to the calcium-binding aYnity of the calmodulin moiety: calmodulin has two classes of calcium-binding sites and site-directed mutations in either high-(K0 d 70 nM) or low (K0 d 11 mM) aYnity sites give rise to constructs in which high-aYnity sites are suppressed to give a monotonic binding curve (K0 d 4.4 mM: cameleon-3) or lowaYnity sites are altered to give an enhanced range over four orders of magnitude of calcium concentration (K0 ds of 83 nM and 700 mM: cameleon-4). The third dimension of modification adds signal tags to the constructs. Nuclear localization tags gave cameleon-2nu and ER localization tags produced yellow cameleon-3er (K0 d 4.4 mM) and cameleon-4er (K0 ds of 83 nM and 700 mM). Tsien’s seminal paper also exemplifies some challenges in the approach: on the one hand, the complexities of permutation and combination of mutant variants and their concomitant properties and on the other hand, the relatively low magnitude of FRET modulation by calcium over a very wide range of concentrations. The subsequent proliferation of family members results from attempts to improve brightness and dynamic range, but at the expense of adding to the combinatorial complexity. Persechini’s second sensor design also concatenated GFPs, MLCK peptide, and calmodulin, though in diVerent order. A calmodulin whose EF hand calciumbinding sites had been reversed in order (CN-CaM) was added to the FIP-CBsm C-terminal to BFP to make FIP-CA (Persechini et al., 1997). This produced a sensor with a monotonic FRET response and a K0 d of 100 nM. Variants with lower aYnities for calcium were obtained by mutating the MLCK calmodulin-binding peptide sequence, rather than the calmodulin calcium-binding sites. As with FIPCBsm, calmodulin binding reduced FRET, the ratio (now expressed as F440/F510) increasing approximately 1.7-fold over the calcium dynamic range. The interaction was markedly pH sensitive in the range 6.5–7.5. This configuration of calmodulin and calmodulin-binding peptide did not lead to later variants and appears to have been an evolutionary dead end. The cameleon family of calcium sensors is shown in Fig. 1.
2. Evolution The EYFP in yellow cameleon-2 and-3 shows an apparent pKa of 6.9, so contains a significant proportion of the protonated species at physiological pH (Miyawaki et al., 1999). The protonated species does not participate in FRET (Habuchi et al., 2002). As pH can vary by several tenths of a pH unit when cells are stimulated; changes in pH would be read as changes in calcium ion concentration. Two adjacent point mutations in EYFP (V68L and Q69k) lower the pKa to 6.1, markedly reducing the pH sensitivity in the physiological range (Miyawaki et al., 1999). Replacing EYFP with EYFP-V68L/Q69K abolished pH sensitivity above pH 6.9 (Miyawaki et al., 1999). This substitution produces yellow cameleon-2.1 (YC2.1; K0 ds for calcium: 100 nM and 4.3 mM) and yellow cameleon-3.1 (YC3.1;
Fig. 1 Schematic depiction of the diVerent classes of genetically encoded calcium sensors. EYFP and EGFP variants for individual sensors are shown to the right, as are the identities of the red-emitting sensors.
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K0 d for calcium: 1.5 mM) with around a twofold diVerence in 528/476 nm emission ratios in calcium-free and calcium-saturating conditions. Recalling that the calcium-dependent signal from FIP-CBsm relied on binding of endogenous calmodulin, an obvious concern would be that YCs would be perturbed by such interactions and also perhaps themselves perturb downstream calcium-signaling pathways. In fact, EC50s for YC2.1 and YC3.1 stimulation of calmodulin-dependent phosphodiesterase were two to three orders of magnitude greater than for calmodulin and the sensors were unperturbed by addition of 3 mM calmodulin. Of course, the YC constructs will buVer calcium inside cells. This was tested by studying the calcium oscillations induced in HeLa cells induced by addition of histamine. At a YC3.1 concentration of 150 mM, calcium oscillations were evident whereas at concentrations greater than 300 mM, oscillations were not seen, though the overall magnitude of the response was little altered. The loss of oscillations suggests calcium buVering. Below around 20 mM, the fluorescent signal was too faint to give acceptable signal-to-noise ratios (Miyawaki et al., 1999). Thus, working YC concentrations in the range 40–150 mM do not substantially perturb calciumdependent signaling mechanisms. Yellow fluorescent proteins, besides being sensitive to pH, are more prone than GFP to photobleaching and to quenching by biological anions such as chloride. Because YFPs show such utility as one of the partners in the CFP/YFP FRET couple, this defect is worth fixing. Mutagenesis by error-prone PCR and expression in Escherichia coli uncovered a mutation to methionine in residue 69 that was much more resistant to chloride quenching than EYFP-V68L/Q69K, twice as resistant to photobleaching, with a pKa of 5.7 rather than 6.1 and of comparable spectral properties including brightness (Griesbeck et al., 2001). This YFP is known as citrine, and substituted for EYFP-V68L/Q69K as the FRET acceptor produced the cameleons YC2.3 and YC3.3. These two cameleons express well at 37 C, show a ratio change of around 1.5 to calcium over their dynamic range and are pH insensitive down to around pH 6.5. To demonstrate the utility of YC3.3 in an acidic compartment, it was targeted to the Golgi using an 81 residue N-terminal construct from human galactosyl transferase type II. The sensor was saturated when expressed in the Golgi, suggesting high resting levels of free calcium concentration in this cellular compartment (Griesbeck et al., 2001). The CFP/citrine couple was also used in an ER-targeted sensor, Cameleon D1ER. Here, the rationale was to design a sensor based on the M13/CaM-biding pair that would be insensitive to interaction with endogenous calmodulin (Palmer et al., 2004), as had been reported (Hasan et al., 2004; Heim and Griesbeck, 2004). The M13 and CaM were co-mutated to provide a binding pair that would not interact strongly with endogenous calmodulin. Cameleon D1ER has a very wide range of calcium sensitivity with K0 ds of 0.81 and 60 mM, appropriate for ER calcium sensing, and was successfully used in HeLa cells to monitor cytoplasmic and ER calcium simultaneously in conjuction with Fura2 (Palmer et al., 2004). The GFP family of proteins is remarkable in possessing a visible wavelength fluorophore that is formed through an oxidation reaction involving adjacent
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amino acids (Tsien, 1998). Fluorescence develops relatively slowly when the protein is expressed in cells, the process of what is known as maturation taking tens of minutes to hours; maturation is also temperature dependent, oxidation to form the fluorophore being the rate-limiting step. Another potential diYculty with FRETbased probes using the CFP/YFP partners is that maturation of YFP is substantially slower than that of CFP, particularly at mammalian body temperatures (Miyawaki et al., 1999), a very important consideration especially for expression in transgenic mammals. If the YFP partner of the FRET couple matures more slowly than the CFP partner, then the sensors dynamic range is compromised, as mature CFP in a sensor that contains immature YFP will contribute to the 476 nm emission in the absence of 528 nm emission from the same construct, so that the overall population 528/476 emission ratio will be depressed as a function of the proportion of disparately matured sensor constructs (as illustrated by the behavior of YC6.1 discussed below; Evanko and Haydon, 2005). The F46L mutation in YFP greatly accelerates oxidation to the mature fluorophore and four additional point mutations contributed to create a construct that matured two orders of magnitude faster than EYFP from a urea-denatured state (Nagai et al., 2002; Rekas et al., 2002); because of its resulting brightness, this YFP construct was given the name Venus. Venus also has a low pKa (6.0) and low sensitivity to chloride, comparable to citrine in these respects (Griesbeck et al., 2001), though it lacks citrine’s improved resistance to photobleaching. Substitution of Venus for EYFP-V68L/Q69K resulted in a new rapidly maturing yellow cameleon (YC2.12). Bright YC2.12 fluorescence was seen to develop rapidly after ballistic transfection of Purkinje cells in cerebellar slices, though the fold ratio change after depolarization suggests that its dynamic range was not much altered from earlier family members (Nagai et al., 2002). The challenge of improving dynamic range was addressed systematically by altering the orientation of the YFP fluorescence dipole relative to the CFP dipole (Jares-Erijman and Jovin, 2003) to maximize FRET (Nagai et al., 2004). Changes in orientation were achieved by circular permutation (see below, Section II.B.1) of the Venus construct. The YC3.12-based construct with EYFP-V68L/Q69K substituted by circularly permutated Venus with a new N-terminal at Asp-173 (termed YC3.60) showed the largest increase in fluorescence emission ratio dynamic range between calcium free and calcium-bound forms in vitro: around 6.6-fold compared to 2.1-fold for YC3.12. This large improvement in dynamic range was verified by expression of each the two sensors in HeLa cells and challenge with ATP to raise cytoplasmic free calcium levels (Nagai et al., 2004). This study also illustrates the important point that altering the properties and conformation of the FRET partners at the N- and C-terminals of the sensor can also alter the apparent calcium activation characteristics of the calmodulin-M13 inner pair as measured by FRET. YC3.60 showed a monotonic increase with calcium concentration, as would be expected from a construct based on the monotonically increasing cameleon-3 (Miyawaki et al., 1997), but the apparent dissociation constant for YC3.60 is 0.25 mM, compared to 4.4 mM for cameleon-3. YC2.60, based on cameleon-2,
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has a single high-aYnity K0 d of 40 nM, compared to the two K0 ds of 70 nM and 11 mM of cameleon-2 (Miyawaki et al., 1997). YC4.6 has K0 ds of 58 nM and 14.4 mM, compared to K0 ds of 83 nM and 700 mM in cameleon-4. The YCX.60 series of cameleons show the rapid maturation and low pH and chloride sensitivities of their Venus forbears, YCX.12, and are the best performing native M13based cameleons to date. Both citrine and the circularly permutated Venus (cpv) of the YCX.60 series were used as alternative acceptors in a further series of cameleons based on Cameleon D1ER (Palmer et al., 2006). Computational design of novel M13 and CaM-based binding pairs led to Cameleons D2, D3, and D4 and D2cpv, D3cpv, and D4cpv, oVering a wide range of calcium aYnities, good sensor dynamic range (the cpv series comparable to YC3.60), and insensitivity to endogenous calmodulin. This cameleon set showed good performance in reporting cytoplasmic and mitochondrial calcium concentrations in HeLa cells and peri-plasmalemmal calcium concentrations in hippocampal neurones when localized with the appropriate targeting sequences. ECFP/EYFP-based cameleons require excitation at near-UV wavelengths. It would be convenient to have FRET-based calcium sensors that can be excited at visible wavelengths. One possibly solution is to use a FRET couple in which GFP is paired with a red fluorescent protein. GFP-like red fluorescent proteins are found in corals (Baird et al., 2000; Miyawaki et al., 2003b). However, they are less tractable than GFP and its variants as they oligomerize, mature very slowly via a green-emitting intermediate and in general, show low extinction coeYcients and quantum yield (Miyawaki et al., 2003b). A GFP/RFP cameleon has been developed using a DsRed variant—a tandem dimer mutant (Yang et al., 2005). The maturation rate is tens of hours and the emission ration change is less than 1.2-fold when cells expressing the sensor are challenged with ionomycin (Yang et al., 2005).
3. Changing the Sensor Mechanism 1 Solution NMR showed that the calmodulin-binding peptide of calmodulindependent kinase kinase (CKKp) has a diVerent relation to the two lobes of calmodulin than M13 peptide (Truong et al., 2001). The structural modeling suggested that the peptide might be concatenated in a recombinant construct between the N- and C-terminal lobes of calmodulin. Calculations suggested that if ECFP and EYFP-V68L/Q69K were attached to the N- and C-terminals of the split calmodulin, then the distance between the fluorophores when calcium was bound and the calmodulin interacting with its binding peptide might be less than ˚ , rather than the 50–60 A ˚ in M13-based YC2.1. Given the sixth power depen40 A dency of FRET on distance between fluorescent dipoles (Jares-Erijman and Jovin, 2003), this approach promised an improvement of the dynamic range of the ratio of fluorescence emission. The splitting of the N- and C-domains of calmodulin in this construct (termed YC6.1) led to a monotonic calcium-binding curve with a K0 d of 110 nM, in some respects more suited to measurement of smaller changes in intracellular free calcium concentration. While in the event, YC6.1 showed a more
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modest fold emission ratio change than predicted (2.1 vs. 1.4 for YC2.1 in parallel experiments), the twofold change was expressed over a narrower range of calcium concentrations (0.05–1 mM) in the physiologically relevant cytoplasmic range. YC6.1 of course suVers from the pH and chloride sensitivity and the slow maturation of its EYFP-V68L/Q69K fluorophore that we discussed above. Replacing EYFP-V68L/Q69K with Venus (Evanko and Haydon, 2005) gives the sensor VC6.1 (Venus cameleon 6.1: the nomenclature is confusing and unhelpful, given that the Venus CaM–M13 cameleons are known as YC2.12 and YC3.12). VC6.1 shows a emission ratio change of around 2.1-fold between zero and saturating calcium concentrations. Thus, as with substitution with Venus for EYFP-V68L/ Q69K to produce YC2.12 from YC2.1, dynamic range is not much altered, while improvements in maturation and pH and chloride sensitivity are obtained. It would be logical to develop a YC6 sensor that contains the circularly permutated Venus used in YC2.6 and YC3.6 (Nagai et al., 2004); this would be predicted to much improve the ratio dynamic range. Small improvements in dynamic range for YC6.1 and VC6.1 can be obtained by excluding from analysis cells that express a low resting YFP/CFP ratio (Evanko and Haydon, 2005): the authors very reasonably suggest that this screens out cells in which the YFP partner is less-mature relative to its CFP pair.
4. Changing the Sensor Mechanism 2 One potential disadvantage of calmodulin-based sensors is that calmodulin is a near-ubiquitous protein with many binding partners. It is possible that calmodulinbased sensors may suVer interference from binding partners when expressed in the cytoplasm or other cellular compartments. While there is no direct evidence to support this conjecture, it is nonetheless true that performance in vivo does not always mirror the sensor properties demonstrated in vitro (Hasan et al., 2004; Heim and Griesbeck, 2004). With this potential pitfall in mind, a sensor has been developed based on troponin C, a calcium-binding protein and close homologue of calmodulin that is, however, expressed only in muscle. The approach was to concatenate TnC with CFP and citrine (Heim and Griesbeck, 2004). While developing these CFP–TnC–citrine sensors, a variant strategy was pursued to concatenate TNI, a TnC-binding partner, alongside TnC by analogy with the M13 binding partner of calmodulin in the classical cameleons; this was unsuccessful. The constructs showing the greatest change in FRET between calcium-free and calciumbound forms contained a chicken skeletal muscle TnC with an N-terminal 14 residue truncation, TN-L15, and a human cardiac TnC, TN-humTnC. TN-L15 showed a 140% change and TN-humTnC a 120% change, measured in the absence of magnesium ion. At physiological (1 mM) magnesium concentrations, the dynamic ranges were 100% and 70%, respectively. Apparent dissociation constants were 470 nM for TN-L15 and 1.2 mM for TN-humTnC. The TnC EF hand calciumbinding sites in TN-L15 were mutated to give K0 ds of 300 nM and 29 mM. The pH sensitivities were similar to the other CFP/citrine-based sensors, with a reduction in
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dynamic range below pH 6.8 and little eVect in the physiological range pH 6.8–7.3. Calcium oV rates were similar to or slightly faster than that of YC2.3 (Heim and Griesbeck, 2004). The TN-L15 sensor was targeted to the plasma membrane using GAP43, Ras, or Synaptobrevin. In direct comparison with YC2.1 and YC 3.3, it showed markedly greater sensitivity and no diminution of dynamic range. Mutations to EF hands III and IV and substitution of citrine with a circularly permutated variant, Citrine-cp174 produced a TnC-based sensor that showed no magnesium dependence, a fourfold dynamic range and a K0 d of 2.5 mM —TN-XL. TN-XL has a very fast oV rate with a dominant component with a time constant of 142 ms (Mank et al., 2006). Expressed in Drosophila under a UAS/Gal4 neuronal promoter, it showed response times to calcium signals at the neuromuscular junction significantly faster than other sensors—YC2.0, YC3.3, Inverse Pericam, G-CaMP1.3, and G-CaMP1.6. Further mutagenesis and rearrangement of the TnC domain gave a higher aYnity variant, modestly named TN-XXL, that was capable of long-term monitoring of individual neuronal responses in flies and mice (Mank et al., 2008).
B. Camgaroos
1. Circular Permutation of EYFP Remarkably perhaps, the beta-can that surrounds the cyclized and oxidized fluorophore is amenable to circular permutation, by which is meant the insertion of a peptide linker between N- and C-terminals of the protein and the creation of a new N- and C-terminal pair elsewhere in the sequence, in the loops that connect the component beta-sheets and in the beta sheets themselves (Baird et al., 1999). As we have seen, circular permutation of Venus led to YC2.60 and YC3.60, the two cameleons with the largest emission ratio dynamic range (Nagai et al., 2004). The discovery that N- and C-terminals of EYFP could be rearranged prompted the discovery that a calcium sensor could be fashioned by insertion of calmodulin within EYFP itself. Xenopus calmodulin was inserted between residues 144 and 146 of each of ECFP, EYFP, and EGFP. Each of these constructs was a calcium sensor, with the EYFP insertion giving the largest calcium response. In calciumfree conditions, the construct absorbs predominantly at 400 nm, while in calciumsaturating conditions, the dominant absorption peak is at 490 nm. The 400-nm absorption is due to the protonated form of EYFP and the 490-nm absorption to the unprotonated form. As discussed in Section II.A.2, in EYFP, the protonated species is not fluorescent (Habuchi et al., 2002), so the excitation spectrum shows a single peak at 490 nm and both the excitation and emission spectra are strongly dependent on calcium concentration, with around an eightfold increase in emission intensity at saturating calcium concentrations. Calcium binding was monotonic with an apparent dissociation constant of 7 mM. Calcium binding clearly shifts the proportion of protonated and unprotonated forms at constant pH, so the pKa’s for the two forms are diVerent: 10.1 and 8.9, respectively. Continuing the whimsical tradition, this calcium sensor is termed Camgaroo-1, because it is yellowish, carries
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a smaller companion (the calmodulin) in a pouch, can bounce high in signal and may spawn improved progeny (Baird et al., 1999) The increase in fluorescence intensity after addition of histamine to Camgaroo-1 expressing HeLa cells was a modest 40% and the characteristic calcium spiking activity was almost invisible, so the sensor is not quite as bouncy as its name implies when sensing cytoplasmic free calcium; however, addition of ionomycin caused an overall sevenfold increase in fluorescence. The modest increase observed in response to histamine is almost certainly due to the 7 mM K0 d, high relative to the calcium increase from around 100 nM to 1 mM expected when histamine is added to HeLa cells. Camgaroo-1 does not fold well at 37 C and could not be targeted to intracellular organelles, for example, mitochondria (Baird et al., 1999). In an attempt to live up to another of its attributes, the possibility that it may spawn improved progeny, Camgaroo-1 was subjected to error-prone PCR mutagenesis (Griesbeck et al., 2001); selection of the brightest clone after expression in E. coli revealed a point mutation of residue 69 to methionine. This new sensor, Camgaroo-2, had very similar calcium-binding properties and fluorescence dynamic range as Camgaroo-1, but expressed far more brightly in HeLa cells grown at 37 C. The response to histamine a (5% fluorescence increase) was lower even than for Camgaroo-1, but targeting to mitochondria using the targeting sequence of subunit VIII of cytochrome c oxidase was demonstrated. Mitochondrial calcium increases that raised the resting fluorescence signal by about 70% were demonstrated in response to histamine and subsequent addition of ionopmycin gave an overall 1.5fold increase in fluorescence signal (Griesbeck et al., 2001), lower than that observed with cytoplasmic Camgaroo-2, perhaps because the resting mitochondrial calcium concentration is higher than that of the cytoplasm. Using a similar camgaroo-like strategy, the EF hand calcium-binding site was introduced into EGFP between residues 144–145, 157–158, or 172–173 (Zou et al., 2007). These Ca-G family sensors had extinction coeYcients and quantum yields comparable to EGFP. They operate in the ratiometric mode and with excitation at 398 and 490 nm showed a sensor dynamic range of 1.8 at a 510-nm emission wavelength. Comprising a single EF hand-binding site, the apparent dissociation constants are in the millimolar range (0.4–2 mM) and are, therefore, suitable only for monitoring high calcium environments such as the ER. They are markedly pH sensitive, with a pKa of around 7.5. Expressed in the ER of HeLa and BHK-21cells, they showed modest ratio changes in response to agonists (Zou et al., 2007).
C. Pericam G-CaMP Family
1. Pericams In pursuit of the idea that the clefts introduced into the beta can structure by circular permutation might make the fluorophore more accessible to solution protons and so susceptible to structural changes brought about by reorientation of concatenated peptides, Miyawaki’s group developed the pericam series of
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sensors (Nagai et al., 2001). Circular permutation of EYFP-V68L/Q69K to give an EYFP with Y145 as the N-terminal and N144 as the C-terminal (cpEYFP) produced an EYFP variant that could be concatenated with M13 and calmodulin (bearing the E104Q mutation that conferred a monophasic calcium-binding curve; Miyawaki et al., 1997). The construct with calmodulin at the N-terminal (CaM– cpEYFP–M13) showed no calcium-dependent properties, confirming the finding reported for a cpGFP variant (Nakai et al., 2001), but the opposite concatenation (M13–cpEYFP–CaM) gave a construct that showed threefold brighter 520 nm fluorescence in high calcium media compared to calcium-free media when excited at 485 nm. This construct was given the name pericam (from a circularly permuted YFP and CaM—calmodulin). Pericam was the prototype from which three pericams with enhanced features were developed. Flash pericam has three additional point mutations that confer an eightfold increase in 520-nm fluorescence on calcium binding. Flash Pericam is a single wavelength, nonratiometric indicator with a K0 d of 0.7 mM . Knowing that substitution of phenylalanine at residue 203 in YFP conferred fluorescence on the protonated form, this mutation was introduced into Flash Pericam. The result, Ratiometric Pericam, was a sensor whose emission ratio at 520 nm when excited at 494 nm or 415 nm changes 10-fold between calcium-free and calcium-saturating conditions with a K0 d of 1.7 mM; this excitation ratio sensor is functionally analogous to fura-2 (Grinkiewicz et al., 1985). Further semirandom mutagenesis of Ratiometric Pericam gave a single wavelength construct whose fluorescence intensity at 513–515 nm decreased on calcium binding—Inverse Pericam (K0 d; 0.2 mM). Two advantages of Inverse Pericam are that it is bright and has excitation/emission characteristics similar to fluorescein; the latter advantage it shares with Flash Pericam: these two YFP-based indicators are functionally equivalent to the Fluo-3 and Fluo-4 single wavelength calcium sensors (Gee et al., 2000; Kao et al., 1989; Minta et al., 1989). Expression in HeLa cells showed that Ratiometric Pericam and Inverse Pericam expressed significantly better at 37 C than did Flash Pericam. Ratiometric Pericam gave a 2.5-fold increase in excitation ratio emission after addition of histamine, while Flash and Inverse Pericams oVer a 100% increase and decrease in signal, respectively, with the same agonist. As might be expected from our earlier discussion of the camgaroos, the calcium-free and calcium-bound forms of all three pericams showed diVerent pKa’s and all three have pH sensitive emissions in the physiological pH range. Miyawaki showed a proof of principle that the excitation ratio-based Ratiometric Pericam can be used in the context of confocal imaging (Shimozono et al., 2002); recent confocal microscopes based on acousto-optical filters oVer turnkey solutions to excitation ratiometric imaging.
2. GCaMPs Single wavelength nonratiometric sensors that use the same sensor strategy as pericams but are based on circularly permutated GFP rather than EYFP were developed at almost the same time as the pericams, their development
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preceding the pericams’ by a matter of months (Nakai et al., 2001). Both the CaM– cpGFP–M13 and M13–GFP–CaM concatenations were tested: only the latter showed significant calcium-sensing properties. Twenty-six variants of the M13– N149cpGFPC144–CaM concatenate were tested and the variant that showed the greatest fluorescence increase in HEK-239 cells after ATP addition was termed G-CaMP (presumably for green fluorescent calmodulin protein). In HEK-239 cells, G-CaMP gave a 1.5-fold increase in fluorescence in response to ATP and a fourfold increase in response to ionomycin. G-CaMP has very similar fluorescence parameters to Flash Pericam, with an excitation maximum at 489 nm, an emission maximum at 509 nm and a 4.5-fold increase in fluorescence on calcium binding (cf. eightfold for Flash Pericam). The apparent dissociation curve was monotonic, with a K0 d of 0.24 mM. As with the camgaroos and pericams and for the same reasons, the sensor signal is strongly pH dependent in the physiological range. The association time constant for calcium binding was strongly calcium dependent and varied from 250 ms at low calcium concentration to 2.5 ms at higher concentrations; the dissociation time constant was 200 ms. G-CaMP expresses poorly at 37 C. G-CaMP-expressing smooth muscle showed a response to rapid depolarization of around 50%, with a time course comparable to that previously measured with Fluo-3. Carbachol addition gave a 2.5-fold increase. pH was monitored in these experiments and did not change (Nakai et al., 2001). This first GCaMP family member, later designated GCaMP1, had very weak fluorescence when expressed at physiological temperatures compared to GFP itself. This was addressed by introducing two mutations V163A and S175G that were known to improve the temperature-dependent maturation of GFP to give a variant known as G-CaMP1.6 (Ohkura et al., 2005); this increased brightness about 40-fold. However, these modifications did not lead to adequate maturation above 30 C. The G-CaMP construct was subjected to error-prone PCR mutagenesis and the clones fluorescing most brightly at 37 C were selected (Tallini et al., 2006). The two new mutations in the brightest clone were identified (D180Y and V93I), but it also turned out that the RSET leader sequence that had been added to facilitate purification of the expressed protein was essential for thermal stability at 37 C. This construct, GCaMP2, is around 200 times brighter than G-CaMP1 at 37 C (with an extinction coeYcient at 487 nm of 19,000 and a quantum yield of 0.93 with emission at 508 nm) and shows the same four- to fivefold increase in fluorescence a saturating calcium concentrations when compared to calcium-free conditions. Though not reported, it should be assumed that this sensor remains pH-sensitive. GCaMP2 was expressed using tissue-specific promoters in transgenic animals and calcium transients were detected in granule cells in cerebellar slices (Diez-Garcia et al., 2005) and in isolated heart in vitro and in adult and embryonic heart in vivo (Tallini et al., 2006). Some insight into the sensor mechanism of GCaMP2 is aVorded by its crystal structure (Akerboom et al., 2009; Wang et al., 2008). Even so, in HEK293 cells, GCaMP2 fluorescence is still 100-fold lower than GFP itself (Tian et al., 2009). HEK293 cell medium-throughput screening assays
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were used to identify brighter GCaMP2 mutants; attention was also paid to improving the sensitivity to small calcium changes through mutations of the CaM EF hands and of the M13/CaM interaction domains. The upshot was GCaMP3, with a dynamic range of 12, due to a twofold decrease in calcium-free fluorescence and a 1.5-fold increase in calcium-saturated fluorescence relative to GCaMP2, and a K0 d of 0.66 mM (Tian et al., 2009).
3. Cases 12 and 16 The Case (presumably Calcium sensor) constructs were developed by analyzing the linker sequences between M13 and cpEYFP/GFP and cpEYFP/GFP and calmodulin and the three key residues 148, 145, and 203 in the pericams and G-CaMPs (Souslova et al., 2007). Based on this analysis, constructs were made containing the G-CaMP linker sequences and the cpEYFP derived from Ratiometric Pericam. Nine point mutants were made with alterations in both the linker sequences and in the three key residues within cpEYFP. As expected, combinations of Asp148 and Phe203 produced ratiometric indicators akin to Ratiometric Pericam, while Asn or Glu at residue 148 combined with Phe203 had a single excitation peak at 490 nm. The Glu148/Thr145 and Glu148/S145 variants showed a 14.5-fold increase in 490-nm fluorescence between calcium-free and calcium-bound forms. The E148/S145 variant of these pericam-G-CaMP hybrids was optimized for folding at 37 C using error-prone PCR, resulting in a variant with a 12-fold dynamic range named Case12. Substituting Thr for Ser at the 145 position of Case12 gave Case16, with a 16.5-fold dynamic range. The apparent dissociation constant for both Cases 12 and 16 was 1 mM. Like the pericams and G-CaMP sensors, the calcium-bound forms of Cases 12 and 16 (pKa 7.2)—and thus their fluorescence—are aVected by any changes in pH within the physiological range.
III. Applications of Genetically Encoded Sensors A. Targeting to Subcellular Locations Low molecular mass fluorescent calcium sensors do make their way to intracellular compartments (Silver et al., 1992) and can be used to measure calcium there, but they are diYcult to target precisely (Varadi and Rutter, 2002b). One of the two major advantages of genetically encoded calcium sensors is that chimeric constructs and signaling tags can target them specifically to subcellular locations. Methods to achieve some of these specific localizations had already been developed for GFP itself and for the calcium sensor aequorin (De Giorgi et al., 1996). The ability to target cameleons YC-3er and YC-4er was demonstrated in the study in which cameleons were first described (Miyawaki et al., 1997).
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1. Endoplasmic Reticulum ER calcium concentrations have been measured using low molecular mass calcium sensors and with aequorin (Solovyova and Verkhratsky, 2002), but it seems fair to say that the cameleon-based sensors (YC-3er and YC-4er) have given the best estimates of ER calcium concentration and turnover (FoyouziYoussefi et al., 2000; Graves and Hinkle, 2003a,b; Varadi and Rutter, 2002a; Yu and Hinkle, 2000). In summary, cameleon-based indicators have presented a picture of the ER as an organelle with resting calcium concentrations in the range 250–600 mM and a very active calcium turnover that depends very heavily on the activity of the SERCA ATPase (Demaurex and Frieden, 2003). Transgenic YC3.3er has been engineered to give tissue-specific expression in mouse pancreatic beta cells (Hara et al., 2004). The interpretation of calcium changes in the ER measured by cameleon indicators is tempered by the finding that pH changes within the organelle may interfere with estimates of dynamic calcium concentration (Varadi and Rutter, 2004). Improved sensors for ER calcium are now available (Palmer Amy et al., 2004; Zou et al., 2007).
2. Mitochondria Mitochondrial targeting of recombinant aequorin was achieved using the N-terminal presequence of subunit VIII of cytochrome oxidase (Rizzuto et al., 1992). The same targeting strategy was used to locate ratiometric pericam within mitochondria (Robert et al., 2001) and to show that the pericam tracked beat to beat calcium changes in cardiomyocytes, just as did aequorin. Cameleon probes targeted to mitochondria were eVective only at very low expression levels (Arnaudeau et al., 2001). In a comparison of mitochondrially targeted cameleon (mtYC2), camgaroo-2, and Ratiometric Pericam (Nagai et al., 2001) in HeLa cells, it was found that Ratiometric Pericam was the most reliable and faithful of the sensors (Filippin et al., 2003). Mislocalization and poor expression of the mitochondrially targeted YC2 sensor could be improved by inserting a tandem repeat of the subunit VIII presequence as the targeting sequence (2mtYC2) (Filippin et al., 2005). 2mtYC2 was used successfully to demonstrate calcium handling by skeletal muscle mitochondria during contraction (Rudolf et al., 2004). Insertion of a tandem targeting repeat was an ineVective strategy for the preferred citrine or Venus variants (Filippin et al., 2005), but in contrast, the D2cpv, D3cpv, and D4cpv cameleons (Palmer et al., 2006) functioned well as mitochondrial calcium sensors when targeted with the cytochrome oxidase tandem repeat (Palmer et al., 2006). These constructs are now the recommended genetically encoded mitochondrial calcium sensors. An recent overview of calcium sensor approaches in mitochondria is available (Pozzan and Rudolf, 2009).
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3. Peroxisome Cameleon D3cpv was furnished with a modified peroxisome localization sequence (D3cpv-KVK-SKL) to monitor calcium concentrations in this organelle in HeLa cells in response to agonists or depolarization (Drago et al., 2008).
4. Golgi The Citrine cameleon YC3.3 has been expressed in the Golgi using an 81 residue N-terminal sequence from human galactosyl transferase type II (Griesbeck et al., 2001); it was saturated, oVering no useful information but that the Golgi has a very high resting calcium concentration.
5. Plasma Membrane Sub-plasmalemmal calcium concentrations may diVer from those in bulk cytoplasm. Localized calcium concentrations around secretory vesicles were shown to be higher than those in cytoplasm by using a phogrin chimera to target YC2 to secretory vesicle membrane (Emmanouilidou et al., 1999). A number of targeting strategies have proved successful in localizing sensors to the plasma membrane. The cpVenus cameleon YC3.60 has been targeted using a Ki-Ras chimera (Nagai et al., 2004). The TN-L15 sensor localized to the plasma membrane as GAP43, Ras, or synaptobrevin chimeras (Heim and Griesbeck, 2004). Localization can also be achieved with a myristoyl/palmitoyl N-terminal tag (Zacharias et al., 2002), an approach that was used with the cameleon D series (Palmer et al., 2006). A chimera of GCaMP2 and synaptotagmin (SyGGCamp2) has been used to monitor synaptic calcium signals, in this case in vivo in zebrafish (Dreosti et al., 2009).
B. Tissue-Specific Expression The other major advantage of genetically encoded calcium sensors is tissuespecific expression in intact organisms.
1. YC2.1 The first transgenic tissue-specific expression of genetically encoded calcium sensors was demonstrated in plants. YC2.1 was expressed in Arabidopsis guard cells of the leaf, first using a CaMV promoter (Allen et al., 1999) and then a guard cell-specific det promoter (Allen et al., 2000), demonstrating that aspects of the calcium-signaling response in guard cells were under diVerential genetic control. YC3.1 was used in transgenic Aradidopsis plants to visualize calcium signals in the pollen grain (Iwano et al., 2004).
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YC2.1 was expressed transgenically in Caenorhabditis elegans pharyngeal muscle under the control of the pharyngeal-specific myo-2 promoter (Kerr et al., 2000) and tracked calcium changes during pharyngeal pumping; YC3.1 tracked temporal changes more faithfully than YC2.1, being the faster sensor, but YC2.1 tracked calcium changes to basal level more faithfully than YC3.1, as might be expected from its lower K0 d. Expression of YC2.12 in C. elegans touch neurons under the control of the mec-4 promoter identified a role for specific ion channels in the touch response (Suzuki et al., 2003). The UAS/Gal4 tissue-specific expression system was used to express YC2.1 in a subset of the antennal lobe projection neurones of Drosophila in order to study odorant responses in the antennal lobe and mushroom body calyx in vivo (Diegelmann et al., 2002; Fiala et al., 2002). Odorant-specific patterns of neuronal excitation were seen in both the antennal lobe and the calyx. In the former, the EYFP/ECFP emission ratio changes were 1.23 0.23% (mean and sem) and in the latter 0.6 0.06%. In the antennal lobe, the changes in sensor signal were observed in spatially restricted regions of around 10–30 mm diameter, the size of individual glomeruli. These very small changes were nonetheless reproducible, with distinct and reproducible patterns of activity from fly to fly associated with diVerent odorants. The same UAS/Gal 4 technology was used to express YC2 in neurones of larval Drosophila (ReiV et al., 2002) to the evolution of calcium signaling in presynaptic terminals innervating larval muscle. A 28% emission ratio change was measured in vivo during spike train stimulation of the neuromuscular junction and signals of this magnitude could be resolved in single synaptic boutons; there were no detectable diVerences in neuromuscular junction physiology between wild-type and transgenic larvae. This study illustrates the point that targeted expression of genetically encoded sensors in individual neurones is for some applications superior to the use of low molecular mass synthetic calcium indicators, as the specificity of expression more than compensates for the loss of brightness. In a similarly mature use of YC2.1 sensor technology coupled to UAS/Gal4 transgenic expression, neuronal calcium measurement coupled with electrophysiology was used to identify thermosensory neurones in the larval nervous system in vivo (Liu et al., 2003). Changes in emission ratio of 10–50% were associated with heating and cooling. A functional map of thermosensory neurones was generated and it was found that neurones with diVerent temperature responses were anatomically segregated. YC2.1 was also used in zebrafish to record the behavior of Rohon-Beard (RB) neurones during the fish’s escape response (Higashijima et al., 2003). This careful study started with transient expression of the YC2.1 transgene in the RB neurones to show proof of principle before generating transgenic lines in which the calcium signals in the RB neurons could be correlated with the escape response in conscious fish.
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2. YC3.3er YC3.3er (the citrine-based sensor) was expressed in the beta cells of transgenic mice under the control of the mouse Insulin 1 promoter (Hara et al., 2004). The sensor signal could be detected in isolated pancreatic islets and addition of thapsigargin or carbachol gave the expected decrease in the 535/485 emission ratio.
3. Camgaroos and Inverse Pericam UAS/Gal 4 expression was used to create transgenic Drosophila that expressed camgaroos-1 and-2 in the mushroom bodies of adult brain (Yu et al., 2003). Dissected fly brains were used. Camgaroo-2 fluorescence in the mushroom bodies was much more intense than that of camgaroo-1, but the camgaroo-1 emission ratio signal on potassium depolarization was more than double that of camgaroo-2 (38% vs. 14% in the mushroom body lobe and 83% vs. 28% in the mushroom body itself ). It was shown that these increases were not due to changes in pH. Application of the putative mushroom body transmitter, acetylcholine, causes ratio changes of a few percent. In this setting, camgaroo-2, although brighter, showed substantially lower ratio changes than camgaroo-1; it also underwent significantly faster photobleaching. Inverse pericam is an intensity-coded sensor that decreases its fluorescence as calcium increases. Addition of DsRed2 to the C-terminal of inverse pericam produces a ratiometric indicator whose 615/510 nm emission ratio increases as calcium increases. This indicator (DsRed2-referenced inverse pericam (DRIP)) requires dual excitation and dual emission optics (Shimozono et al., 2004). The DsRed2 fluorescence is a passive, calcium-independent signal that is proportional to the concentration of the sensor and helps control for alterations in overall fluorescence intensity due for example to movement artifacts. DRIP was expressed transgenically in worms under the control of the myo 2 promoter that is specific for pharyngeal muscle. Ratio changes of 30–40% were measured in worms undergoing fast pharyngeal pumping. After screening six sensors (flash pericam, inverse pericam, G-CaMP, camgaroo-2, YC2.12, and YC3.12) for calcium sensitivity in stably transfected fibroblast cell lines, the two with the greatest dynamic range (inverse pericam: 40% and camgaroo-2: þ 170%), together with YC3.12 that gave inconclusive results in the fibroblast expression screen but is optimized for expression at 37 C, were used to generate transgenic mice under the control of the TET expression system (Hasan et al., 2004); the TET system allows tissue-specific expression by crossing the TET mice with mice expressing the TET transactivator under tissue-specific control. TET sensor mice were crossed with a line expressing the transactivator under the control of the alphacalmodulin/calcium dependent kinase II (aCamKII) promoter. All mice developed normally. Five highly expressing lines were obtained out of 36 transgenic lines: two YC3.12, two camgaroo-2, and one inverse pericam. Expression patterns in brain slices and excised retina were analyzed by two-photon microscopy. They appeared to
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be mosaic, not mapping directly to the known patterns of aCamKII expression. Neocortical expression could also be imaged through the thinned skull in anaesthetized mice. Two photon fluoresence recovery after photobleaching suggested that as much as half the fluorescence signal was immobile and this together with punctuate staining patterns suggested that this immobile sensor fraction might be due to interaction between the M13 and CaM moieties of the sensors and their normal cellular targets. Cellular and synaptic stimulation of pyramidal neurones in cortical slices using sharp and patch microelectroded gave 5–10% increases in 535 nm fluorescence using wide field imaging and around 20–100% for camgaroo-2 and 30% for inverse pericam using two photon imaging. In the retina, a ganglion cell subset was strongly labeled in YC3.1-expressing mice, but no lightevoked responses were detected. In camgaroo-2 expressing lines, bleaching occurred in the retina too quickly for measurements to be made. In one inverse pericamexpressing mouse, 7 of 12 ganglion cells tested showed a transient decrease in fluorescence attributable to a calcium increase in response to light. Sensors were imaged in the olfactory bulb in vivo using wide field microscopy. Camgaroo-2 expressing mice showed a 1–3% increase in response to odors, while inverse pericam gave 8% decrease. Each distinct odor evoked a unique pattern of activity, similar odors evoking similar patterns. This thoughtful study established four main facts: around half of the transgenically expressed cameleon family sensor was immobile; this reduced sensitivity and made quantitation of the calcium signals impossible; nonetheless, it was possible to observe patterns of neuronal activity; YC3.12 was not an eVective transgenic sensor. The study also reports unpublished experiments in which transgenic mice expressing YC3.0 under the control of a b-actin promoter gave only 1–2% ratio changes during wide filed imaging in cerebellar slices. The high proportion of immobile sensor in transgenic animals remains for the moment inexplicable—it was not seen in the stably transfected fibroblast lines.
4. GCaMP G-CaMP (Nakai et al., 2001) was expressed in mice under the control of a smooth muscle myosin heavy chain promoter and was expressed in vascular and nonvascular smooth muscle (Ji et al., 2004). The signatures of inotropic (ion channel) and metabotropic (InsP3-mediated) postsynaptic signaling could be distinguished in single excised smooth muscle cells. In a set of experiments strikingly parallel to those with YC2.1 (Diegelmann et al., 2002; Fiala et al., 2002), but using two photon imaging, G-CaMP was expressed in a subset of projection neurones in Drosophila antennal lobe to demonstrate that diVerent odorants activated specific patterns of glomeruli (Wang et al., 2003). Individual glomeruli are diVerentially sensitive to a given odorant and more are recruited as the odorant concentration is increased. Increases of fluorescence of up to 50% (at 525 nm) were measured in responsive glomeruli.
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Transgenic expression of GCaMP2 has been achieved in mouse heart (Tallini et al., 2006). The TET system was used: the GCAMP2 sequence was placed downstream of a weakened a myosin heavy chain promoter (aMHC) and seven tetO enhancer sequences to permit suppression of gene expression using doxycycline. These mice were crossed with others with a hemizygous aMHC-tetracycline transactivator allele. The doubly transgenic mice expressed GCaMP2 only in the heart. Doxycycline suppression of the transgene was essential, as mice constitutively expressing GCaMP2 from birth showed markedly enlarged hearts, a phenotype comparable to that seen in mice overexpressing calmodulin. This phenotype was avoided entirely by administering doxycycline in utero and until 13–15 weeks postpartum. Subsequent removal of doxycycline for up to 6 weeks led to no detectable cardiomegaly. Robust GCaMP signals were present 4 weeks after doxycycline removal. Striking wide field fluorescence images of cardiac calcium transients in whole mouse heart beating at up to 300 beats/min were obtained with anaesthetized, ventilated open-chested mice, the first to be recorded under wholly physiological conditions with the heart under normal load. As expected sympathetic stimulation with isoproterenol markedly increased the calcium signal and also increased end diastolic calcium concentration. Signal-to-noise ratios were good and it was possible to record very clean signals from a single pixel of the 100 100 pixel imaging array (tens of microns). Using a photodiode array in isolated perfused heart, signals from a membrane potential sensitive dye and from GCaMP2 were acquired simultaneously. Association and dissociation kinetics of calcium were rapid (t ¼ 14 and 75 ms, respectively) and unaltered in vivo. Comparison with a fast calcium dye Rhod2 nonetheless showed that the rise and decay times of the GCaMP2 signal in beating heart was around 45% slower, but with a three times greater dynamic range. Calcium sparks could not be observed in isolated ventricular myocytes expressing GCaMP2. GCaMP2 imaging in open-chested embros from embryonic day 10 allowed the analysis of the development of the atrio-ventricular node conduction pathway. GCaMP2 fused to synaptotagmin localizes to synaptic boutons. It reports the location of synapses in zebrafish in vivo and shows a linear response over a wide range of action potential frequencies (Dreosti et al., 2009). It can report spiking frequencies in optic tectum; it also reports activity in the graded synapses of retinal bipolar cells. GCaMP2 has also been used to map functional connections in the C. elegans nervous system (Guo et al., 2009). Connections can be mapped grossly, but the sensor’s signals are too weak to distinguish direct from indirect connections.
5. TN-L15, TN-XL, and TN-XXL A cerulean version of TN-L15, cerTN-L15, was used to create a transgenic mouse line that expressed the sensor widely in brain, especially in the neocortex and hippocampus (Heim et al., 2007). Calcium changes resulting from two to three action potentials could be resolved and calcium responses in spiny dendrites of
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pyramidal cells could be detected after puYng on glutamate, an excitatory neurotransmitter (Garaschuk et al., 2007; Heim et al., 2007). TN-XL was expressed using the UAS/Gal4 tissue-specific expression system in Drosophila neuromuscular junction (Mank et al., 2006). Its rapid oV rate for calcium made it significantly better at tracking calcium changes than its counterparts. TN-XXL showed improved sensitivity and long term-stability in sensing calcium signals from fly neurones; in mice, tuning curves for orientation-specific neurones in visual cortex could be monitored repeatedly over timescales of days or weeks (Mank et al., 2008).
6. Comparison of the Performance of Genetically Encoded Calcium Sensors Though progress in the field has been periodically reviewed (Barth, 2007; Garaschuk et al., 2007; Griesbeck, 2004; Mank and Griesbeck, 2008), few studies have systematically compared the performance of diVerent genetically encoded calcium sensors, except to demonstrate the superiority of a novel sensor over its predecessors. I have discussed above (Section III.B.3) the systematic comparisons of camgaroo-1 and-2 when expressed in Drosophila mushroom bodies (Yu et al., 2003) and of inverse pericam, camgaroo-2 and YC3.1 when expressed in mouse brain (Hasan et al., 2004). The performance of GCaMP, inverse pericam, and camgaroo-2 was compared with that of the low molecular mass synthetic indicators X-Rhod-5F and Fluo4FF in apical dendrites of pyramidal cells in hippocampal brain slices from 6- to 7day-old rats transfected using a biolistic approach and maintained at room temperature (Pologruto et al., 2004). Images were obtained using two-photon microscopy. Action potentials were triggered using current injection into the cell body. Under these conditions, X-Rhod-5F and Fluo4-FF could detect calcium changes (signal twice that of noise) in the dendrite due to voltage-dependent calcium channel activation after single action potentials while with the same criterion GCaMP required five action potentials, camgaroo-2, 33 action potentials, and inverse pericam over 20. For comparison, the dynamic ranges (DF/F) for the three sensors under these conditions in vitro was1.8, 2, and 0.25, so the sensitivity of camgaroo-2 was poor despite its larger dynamic range. Power spectrum analysis was used to analyze the fluorescence response during action potential trains at 20 Hz. Most of the power in the frequency analysis of X-Rhod-5F and Fluo4-FF fluorescence was at the fundamental frequency, 20 Hz, indicating that the fluorescence signal tracked each action potential. For the genetically encoded sensors, no clear peak was observed at 20 Hz, indicating that the sensors were too slow to track individual action potentials at this stimulation frequency. It was possible to measure calcium activation curves in situ for the three sensors and thus their apparent dissociation constants by simultaneously measuring calcium concentration using a calibrated X-Rhod-5F signal and the fluorescence signal from the sensor at various levels of stimulation. For inverse pericam (K0 d 0.9 mM) and camgaroo-2 (K0 d 8 mM), these were comparable to those previously reported in vitro; however, GCaMP showed a K0 d (1.7 mM) almost an order of
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magnitude greater than that previously reported in vitro (Nakai et al., 2001). Because the calcium concentration profile of dendritic action potentials is well characterized (Pologruto et al., 2004), there seems little doubt that the calcium dissociation characteristics of GCaMP vary markedly in vitro and in vivo. FRAP studies in dendrites showed that the all three sensors were mobile, with mobilities comparable to GFP itself. This result is quite firmly at odds with that reported in mouse brain (Hasan et al., 2004) and discussed above (Section III.B.3). It may be that dendrites, being relatively free of organelles, mirror better the behavior of the sensors in cytoplasm than cell bodies; it should be noted that punctuate staining was reported in mouse brain (Hasan et al., 2004). It should also be borne in mind that though the observations on mouse brain slices were carried out at room temperature, as were these experiments in rat brain slices, in the mouse study, the sensors had been expressed at body temperature, whereas the biolistically transfected rat brain slices were maintained throughout at room temperature. These data, as the authors point out (Pologruto et al., 2004), demonstrate that the genetically encoded sensors are better-suited to measuring summated neuronal responses after multiple stimuli, not single action potentials, consistent with their reported use to monitor patterns of neuronal activity (Fiala et al., 2002; Hasan et al., 2004; Wang et al., 2003); as it happens, these three studies all described odorant-specific patterns of neuronal signaling. As an addendum to the study, Svoboda’s group also provided in vitro solution X-ray scattering evidence that showed that the calcium-dependent fluorescent signal of GCaMP, as theorized, depends on a coupled structural change in which calcium binding to CaM is closely linked to binding of CaM to M13; in contrast, the calcium-dependent fluorescence signal in camgaroo-2 is solely due to binding to CaM, the M13 peptide paradoxically playing no part in the sensor response (Pologruto et al., 2004). A second comparative study was undertaken at the Drosophila larva neuromuscular junction (ReiV et al., 2005), using an approach previously reported (ReiV et al., 2002). The responses of 10 sensors from the three families to 40 and 80 Hz stimulation of the synaptic bouton were compared. Camgaroos-1 and-2 and flash pericam did not sense calcium changes in the bouton. YC2.0, 2.3, 3.3, TN-L15, inverse pericam, and GCaMP1.3 and 1.6 all showed adequate responses (around 5% on average at 40 Hz and 10–15% at 80 Hz) to pulse train stimuli, but none exhibited dynamic ranges anywhere near comparable to those measured in vitro (ReiV et al., 2005). None was comparable in performance in this system when compared to the later developed TN-XL (Mank et al., 2006). In an echo of the work in rat brain slices, the performance of YC3.3, TN-L15, GCaMP1.6, GCaMP2, YC2.60, YC3.60, cameleon D3, and TN-XL were compared one with another and calibrated against a low molecular mass indicator, Oregon-GreenBAPTA-1 (Hendel et al., 2008). The latter four sensors were around twofold more responsive than their earlier counterparts. None of the sensors were seen to detect single action potentials, though YC3.60 and cameleon D3 could detect two action potentials in succession. None showed the fast temporal response of the low
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molecular mass indicator. A theoretical framework in which to consider the pros and cons of calcium sensors in recording neuronal activity has been adumbrated (Hires et al., 2008). GCaMP1.6 and GCaMP2 were compared in pyramidal cells dendrites in mammalian brain slices transfected ballistically or by electroporation (Mao et al., 2008) under conditions that allowed comparison with first generation sensors (Pologruto et al., 2004). Their performance was not significantly better than GCaMP, even when localized using membrane and cytoskeletal targeting chimeras (Mao et al., 2008). GCaMP3, however, showed substantial gains in sensitivity and discrimination (Tian et al., 2009): overall, the signal-to-noise ratio was much improved and responses in dendrites to single action potentials could be reliably detected. Direct comparison with TN-XXL and cameleon D3 showed that, although brighter, the two FRET sensors gave smaller fluorescence changes and less favorable signal-to-noise ratios. GCaMP3 was also more photostable. After either adenoviral transfection or in utero electroporation, calcium responses in pyramidal neurones could be observed in awake, behaving mice (Tian et al., 2009). Parallel electrical recordings showed that detectable calcium responses were associated with three or more action potentials. Calcium responses were also readily observed in the glomeruli of Drosophila antennal lobe and in sensory neurones of C. elegans, altogether a methodological tour de force (Tian et al., 2009).
IV. Use of Genetically Encoded Calcium Sensors For single cell applications, wide-field fluorescence imaging, spinning disk, or confocal microscopy are appropriate methods. Dual excitation laser scanning confocal imging is achievable (Shimozono et al., 2002). For whole animal applications, particularly in intact brain or brain slices two photon microscopy is recommended, as it reduces tissue damage and oVers improved imaging within tissue (see Chapter 9; Fan et al., 1999). Expression of sensors in cells and tissues, as we have seen, can be achieved by transfection and transgenesis. One advantage of transgenic approaches is that expression can be confined to a specific tissue or cell type, an advantage even if it is excised for imaging. Random expression in a subset of cells can more simply be achieved by using biolistic transfection of excised tissue. Ratiometric sensors (in this context the FRET-based sensors, ratiometric pericam and DRIP) oVer the advantage that the quantitative signal is in theory independent of variations in sensor distribution and concentration within cells (Silver et al., 1992). This allows reliable calibration of the signal in terms of calcium concentration (see chapter 1). Nonratiometric sensors (e.g., GCaMP3) are adequate for determining changes in calcium concentration, for example, when measuring overall spatial and temporal patterns of calcium signaling. Even in these circumstances, caution should be exercised in case the responses are nonlinear,
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especially at low calcium concentration, so that a subset of smaller signals is overlooked (ReiV et al., 2005). In general, genetically encoded calcium sensors are not available commercially, though Invitrogen oVers YC3.60 (http://probes.invitrogen.com/media/pis/ mp36207.pdf). Some can be obtained for noncommercial use from their creators (http://www.tsienlab.ucsd.edu/ and http://cfds.brain.riken.jp/). Or you can make your own using the handbook (Miyawaki et al., 2003a, 2005).
V. Conclusions Genetically encoded calcium sensors have proved valuable in the measurement of calcium concentration in cellular organelles, for the most part in single cells in vitro. Their success as sensors in tissues in vitro and in vivo is qualified. They have also proved valuable in imaging the pattern of calcium signals within tissues, particularly in the poikilotherms, C. elegans, Drosophila, and zebrafish. In homeotherms, the record is largely disappointing, even when tissue is excised and monitored at room temperature (Pologruto et al., 2004). Striking exceptions are the use of GCaMP2 to image calcium-signaling patterns in mouse heart (Tallini et al., 2006) and pyramidal neurones (Tian et al., 2009). For the most part, sensors are still not capable of sensing individual calcium events in single cells when these cells are part of tissue, though single cell responses can be monitored in disaggregated cells (KotlikoV, 2007). Some branches of the calcium sensor evolutionary tree continue to evolve rapidly and the steady progress in optimizing sensor parameters leads to the certain hope that these drawbacks will eventually be overcome by further genetic engineering.
Acknowledgments I thank Jill McKenna for helping with this chapter. Our work is supported by grants from the Wellcome Trust.
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CHAPTER 7
Patch Clamp Methods for Studying Calcium Channels David L. Armstrong, Christian Erxleben, and Jody A. White Membrane Signaling Group Laboratory of Neurobiology National Institute of Environmental Health Sciences NIH, Durham, North Carolina, USA
Abstract I. Introduction II. Rationale III. Methods A. Assembling the Patch Clamp Rig B. Making Pipettes C. Making Seals D. Making Recordings IV. Materials V. Discussion VI. Summary References
Abstract The patch clamp technique, which was introduced by Neher and Sakmann and their colleagues in 1981, has allowed electrophysiologists to record ion channel activity from most mammalian cell types. When well-established precautions are taken to minimize electrical and mechanical fluctuations, current transients as small as 0.5 pA and as brief as 0.5 ms can be measured reliably in cell-attached patches of plasma membrane with a polished glass pipette when it forms a giga-ohm seal with the membrane. In many cases, this is suYcient to watch individual channel proteins open and close repeatedly in real time on metabolically intact cells. No other technique currently provides a more precise or detailed view of the function and regulation of calcium channel gating. If antibiotics are added to the pipette to permeabilize the METHODS IN CELL BIOLOGY, VOL. 99
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membrane underneath to small monovalent cations, thereby allowing the entire cell to be voltage-clamped without disrupting its contents, the integrated activity of all the calcium channels in the surface membrane can be measured.
I. Introduction Calcium ions trigger many fundamental cellular processes by binding to proteins, usually with dissociation constants around 1 mM calcium. However, unlike other second messengers such as cyclic nucleotides, calcium is neither created nor destroyed by biological processes. Therefore, regulating calcium-dependent processes requires moving calcium ions into and out of cellular compartments. Transport proteins that hydrolyze ATP move calcium ions from lower to higher concentrations. They maintain resting cytoplasmic calcium at 100–200 nM by pumping calcium out of the cytosol into the endoplasmic reticulum, the mitochondria, or out across the plasma membrane. By contrast, ion channel proteins, when the channels are open, allow calcium ions to diVuse rapidly down their electrochemical gradient back into the cytosol. Many channels are permeable to calcium because they pass all cations, including calcium, up to a certain size, but most of the current is carried by more prevalent ions such as sodium or potassium. In contrast, some ‘‘calcium-selective’’ channels pass calcium almost exclusively, even in the presence of other cations. There are currently three classes of calcium-selective channel proteins (Table I): three families of voltage-gated calcium channels in the plasma membrane (CaV1-3); two families of calcium-selective channels in the endoplasmic reticulum membrane;
Table I Calcium-selective channels Voltage-activated calcium channels in the surface membrane L type CaV1.1 CACNA1S CaV1.2 CACNA1C CaV1.3 CACNA1D CaV1.4 CACNA1F P/Q type CaV2.1 CACNA1A N type CaV2.2 CACNA1B R type CaV2.3 CACNA1E T type CaV3.1 CACNA1G CaV3.2 CACNA1H CaV3.3 CACNA1I Calcium release-activated channel (CRAC) in the surface membrane STIM-gated ORAI1-3? Ligand-gated calcium channel in the endoplasmic reticulum ITPR1-3 IP3-gated Ryanodine-gated RYR1-3
Skeletal muscle Cardiovascular muscle Endocrine cells, neurons Retina Nervous system Nervous system Nervous system Brain, heart Brain, endocrine cells, heart Brain
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and calcium release-activated channels (CRAC) that mediates store-operated calcium entry (SOCE) across the plasma membrane (Hille, 2001; Hogan et al., 2010). All of these proteins provide channels that allow calcium to diVuse into the cytosol when the channel opens. Each open channel protein has a unique unitary conductance for calcium, ranging from approximately 0.1 to several hundred picosiemens (pS), but the proteins spontaneously cycle between open and closed conformations on a time scale of milliseconds. It is the rates of these transitions rather than the conductances which are regulated by physiological events to control the amplitude of calcium fluxes. Such unitary currents are often diYcult to measure because they are small and individual openings last less than a millisecond. However, the total amount of current crossing the entire surface membrane of a cell at any time is the product of the number of channels (N), the fraction of time they spend in the open state (Po), their unitary conductance (g), and the electrochemical driving force (DV) measured as the diVerence between the voltage across the membrane (Vm) and the ion’s Nernst potential. Even small currents produce physiologically significant increases in intracellular calcium. For example, a current of only 0.1 pA (pA ¼ 10 12 A), which corresponds to 0.1 pC of charge per second, or 300,000 divalent ions per second, will transfer 300 calcium ions each millisecond the channel is open. Such a current, 0.1 pA lasting 1 ms, is just below the current technology of detection with the patch clamp technique. Nevertheless, it would produce a physiologically significant change in intracellular calcium. The calcium ions cannot diVuse on average much farther than a micrometer in a millisecond, so the concentration under the membrane will rise transiently to 0.5 mM, more than double the resting level of calcium. If there was only one such channel that opened for 1 ms in every square micrometer of membrane of a spherical cell with a diameter of 10 mm (volume 0.5 pL; area 300 mm2), then the resulting 30 pA current would almost double intracellular calcium concentration throughout the cell. Action potentials that depolarize cells for tens of milliseconds will have correspondingly larger eVects. Thus, millisecond diVerences in calcium channel kinetics have profound consequences for cell physiology and human health (Erxleben et al., 2006). This calculation also illustrates the danger of expressing recombinant channels in mammalian fibroblasts. Investigators routinely report currents of a few nanoamperes, which even inexperienced investigators can measure with the patch clamp technique. However, in the scenario outlined above, a 3-nA current would represent a 100 higher density of channels and produce 100 larger increases in calcium, which might lead to cytotoxic reactions. In most cases, such recordings are made with exogenous calcium buVers in the cytosol, which not only prevent cytotoxicity but also preclude analysis of physiological regulation of calcium channels by calcium-dependent signaling. In addition, because most calcium channels have a low probability of opening (Po) less than 0.1, the larger current density reflects at least 1000 channel proteins per square micrometer, or more than 10% of the space available with close packing. At this density, there might not be room for each channel protein to be associated with its normal penumbra of
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regulatory proteins, even if their expression was upregulated by the cell to compensate for the increased expression of the channels. Recording the activity of recombinant channels from cells that are dialyzed with simple salt solutions using the conventional ‘‘whole-cell’’ configuration of the patch clamp is also dangerous because in the absence of normal metabolic activity, many calcium channels have conformations from which it is diYcult to elicit robust activity with physiological stimuli. Nevertheless, if the plasmid drives the expression of 100–10,000 times more channels than are normally present, even channels with very little activity might generate quite large currents. We would argue, however, that identifying the mechanisms that halve or double the activity of recombinant channels, which only open for 1 or 2 ms every second, might not be relevant to their physiological functions. Thus, this chapter focuses on the more diYcult and consequently less frequently used techniques of single-channel recording from cell-attached patches and the perforated patch technique for voltage clamping metabolically intact cells.
II. Rationale Calcium signaling can be investigated at many levels. Although the patch clamp method is a very quantitative technique for measuring ion fluxes across the plasma membrane, internal stores of calcium are important sources that the patch clamp technique cannot access. To study calcium release from internal stores through calcium-selective channels, one must use fluorescent calcium indicators or reconstitute the channels from organelles in bilayers. This chapter provides an overview of the patch clamp method for measuring voltage-activated calcium currents across the plasma membrane. We have applied this technique primarily to dissociated mammalian cells in vitro culture, and it has also been adapted to studying neurons in brain slices (Sakmann and Neher, 1995). Very basically, the technique involves the use of an operational amplifier circuit to clamp voltage changes between a wire in the patch pipette and a wire in the bath and the measurement of how much current it takes to hold the voltage constant. When a giga-ohm (GO ¼ 109 ohms) seal is formed between the glass patch pipette and the cell membrane, background current fluctuations can be reduced suYciently to detect picoampere currents. Gigaohm seals are most eVective for stable, low-noise recording when they are in the 10–100 GO range. However, in our experience, most investigators routinely settle for seals in the 1–10 GO range. The methods described below allow us to routinely obtain seals around 50 GO. Unfortunately, they are all necessary for success. When the patch clamp technique was introduced almost 30 years ago (Hamill et al., 1981), it was the only quantitative method available to obtain reliable physiological information about calcium signals in mammalian cells and the proteins that mediate them. Now, however, there are increasingly sophisticated calcium indicators that can be targeted genetically to specific compartments in
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specific cells. In addition, genome cloning, fluorescence confocal microscopy, and structural studies of membrane proteins have advanced to the point where they are better suited than the patch clamp technique for identifying which channel proteins are expressed by specific cell types, how their distribution on and traYcking to and from the cell surface is regulated, and their three-dimensional molecular structures. Nevertheless, the patch clamp remains the technique of choice for investigating the channels’ physiological function and regulation at the molecular level in real time. In the future, genetic manipulation of channel sequence and expression in model organisms and mass spectrometry of posttranslational modifications might begin to supplant even these applications of the patch clamp technique. Three volumes of Methods in Enzymology have already been devoted to the patch clamp method, so it would be impossible to provide a suYciently detailed introduction here that would allow a novice to make recordings without consulting other sources. Scientists with little training in physics and/or membrane physiology should start with the ion channel primer by Aidley and Stanfield (1996) and the technical primers by Molleman (2003) and Ogden (1987). More experienced physiologists could move directly to the compendia by Sakmann and Neher (1995) and by Rudy and Iverson (1992). More specialized topics are covered in subsequent volumes edited by Conn (1998, 1999). If you are creating your own rig from scratch, chapter by Jim Rae and Rick Levis in volume 207 is essential reading.
III. Methods A. Assembling the Patch Clamp Rig To localize contributions to noise, it is important to assemble the rig slowly, piece by piece. There are two general classes of noise to eliminate initially. Sinusoidal fluctuations arise from electronic interference by power sources and from mechanical vibrations. In practice, they are often diYcult to distinguish because they produce oscillations of similar frequency, but both should be eliminated completely. Later, after giga-ohm seal formation, any remaining noise should have much higher frequencies. Some of this noise arises from capacitative coupling between the glass wall of the pipette and the salt solution surrounding it in the bath, which can be minimized experimentally. Remaining contributions to high frequency noise can only be reduced by filtering the signal, which limits how brief an event of a given amplitude one can detect. To begin, run the calibration tests that are specified by the manufacturer on the amplifier while it is sitting on a desk. Then assemble the air table, the faraday cage, the microscope, and the manipulator. They should share a common ground. The lamp of the microscope will require a DC power supply and each of the manipulator’s motors must be grounded separately. Surprisingly, despite all their metal parts, many microscopes are not isopotential and need to be grounded at several points. With the input of the amplifier’s headstage still open and the output filtered
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Mechanical sources Table floating freely Microscope and manipulator tightly secured to table Electrical lines and perfusion tubing lashed tightly to microscope base or table No air drafts blowing on rig Rubber gasket that holds glass capillary in pipette holder in new condition
to 100 Hz, confirm that there are no sinusoidal fluctuations at 10 the highest gain you plan to use. Table II contains a checklist for major sources of noise. Finally, test the pipette holder, metal electrodes, and perfusion system by inserting into the recording chamber a cover slip that has a hemisphere of solidified sylgard elastomer (184, Dow Corning). Pressing a patch pipette into the sylgard allows you to obtain seals of up to 100 GO. Silver wires can be chlorided by dipping them in chlorine bleach for several minutes, then rinsed thoroughly and air dried. Because silver is toxic to cells, the ground wire is connected to the bath through an agar salt bridge.
B. Making Pipettes Commercial programmable pipette pullers are now available, but some experimentation is required inevitably to find the settings that produce pipettes with the desired overall shape: long and narrow tapers for cell-attached patch recordings to minimize capacitance with the bath; and short stubby tapers for perforated patch whole-cell recordings to speed antibiotic diVusion to the tip and minimize access resistance. Handling the capillaries with wet or greasy hands on the portion that is heated contributes to variability, but before pulling the pipettes, both ends of the capillaries should be fire polished gently to increase the longevity of the silver chloride coating on the wire in the pipette holder. Otherwise, the sharp glass edge at the back of the pipette scrapes oV a little silver chloride every time you insert a new pipette. Also make sure the pipette holders and their internal rubber gaskets are designed to hold capillaries of the same outer diameter as your pipettes, or they will not fit snugly. For most low-noise applications, the pipette must be coated with ‘‘Sylgard’’ (Corning 184), an elastomer that is cured with heat, which insulates the pipette walls from the bath solution. The reported curing time for Sylgard is 10 min at
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150 C; however, a brief exposure (< 1 min) to a heating element followed by 10– 20 min at room temperature should be adequate for curing before moving on to shaping of the tip. The Sylgard is most eVective when it is applied thickly, but prevents giga-ohm seal formation if it gets on the tip. Our method of applying Sylgard is illustrated in Fig. 1. We use gravity by mounting the pipette vertically
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Fig. 1 Application of Sylgard. Sylgard (Corning 184), a heat-cured polymer, is applied to patch pipettes after pulling but before polishing to reduce the capacitance between the glass walls and the bath. (A) Sylgard is stored in small 1-ml plastic tubes in the freezer until it is ready for use. Room temperature Sylgard is applied using a 25-gauge hypodermic needle that is bent for ease of application. The needle hub is aYxed to a plastic tube for ease of handling. (B) The pipettes are placed into a hand-made holder that holds the pipette upright and allows it to be positioned easily so that the heating element made of tungsten wire surrounds the tip. (C) The pipette is positioned so that the area from the first narrowing of the glass to the tip is placed above the heating element. (D) Sylgard is applied in consecutive rings or ‘‘donuts’’ starting at the area where the glass first narrows. This lower ring helps protect against noise if your bath height changes between perfusions. (E) Between applications of consecutive rings, apply heat to cure each ring. Remember that heat goes up, so the hottest area near the heating wire is above it. (F) The final ring should also coat the taper of the pipette and can be placed as close as 50–100 nm from the tip. Be careful not to get Sylgard on the tip or on the heating element!
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inside a loop of tungsten wire across the prongs of an outlet plug. We control the heat transiently with a rheostat and observe the pipette through an inexpensive, low power, dissecting scope mounted horizontally on a boom arm. The Sylgard is precured to a viscous consistency at room temperature when it is first made and then stored in small 1 ml aliquots in the freezer. We apply it with a bent hypodermic needle by taking up a dollop onto the needle, touching the dollop to the pipette, and slowly winding it on to the glass by turning the pipette from below. Care must be taken to avoid touching Sylgard to the heating/curing wire, or the Sylgard vapors will quickly coat the tip. You will know when you get Sylgard too close to the tip because when you try to polish it, the Sylgard contracting around the thin glass walls at the tip literally wrinkles the glass. Everybody has their own favorite glass that they believe forms the tightest seals, but their relative intrinsic noise can be tested empirically using the Sylgard hemispheres. The lowest noise is produced by quartz glass capillaries (Levis and Rae, 1993), but they require a special laser puller to create the pipettes. On real cells, the size and shape of the tip also influence success. It is extremely diYcult to make pipettes that will routinely make > 10 GO seals when you cannot clearly see the tip while you polish it. An extra long working distance (ELWD) objective with at least 40 magnification is essential but they are expensive, so they must be mounted below the polishing element that is heated to prevent cracking the lens from repeated heating. Our polishing strategy is illustrated in Fig. 2. We find that it is also critical to melt a small bead of the pipette glass onto the apex of the heating element, usually a small loop of platinum wire, presumably because it prevents the tip from being coated with metal. We find that pipettes with bullet-shaped tips and initial resistances between 3 and 5 MO make the best seals. Filling the pipettes also requires some attention to detail. All pipette solutions should be filtered through 0.2 or 0.45-mm filter disks but do not use filter disks prepared with wetting agents. To avoid bubbles and washing the dirt inside the capillaries down into the tip, both of which reduce seal success, one must first fill the tip separately by immersing it in a small vial of pipette solution and allowing the first 20–50 mm to fill by capillary action. Then the rest can be backfilled. Usually bubbles are visible to the naked eye, and they can be removed by gently flicking the pipette while it is held between the thumb and the forefinger. C. Making Seals To reliably get seals over 10 GO, all the precautions in Table III are important. In addition you have to be fast. It should take only 3–5 min from filling the pipette to touching down onto the cell, including mounting the pipette in the holder, manipulating it into the chamber, zeroing out the oVset, measuring the resistance, finding it in the microscope, and manipulating it just above the cell without touching the bottom or another cell. This takes practice. It also helps to run a little solution through the chamber before lowering the pipette into the bath to clear any debris that accumulates on the surface. To minimize debris from collecting on the patch
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Fig. 2 Fire-polishing pipettes. Fire polishing the tip allows the user to narrow the tip opening to gain the desired shape and resistance. (A) The pulled pipettes are placed horizontally in a 2D holder mounted on the stage of an inverted microscope with a long working distance objective 40. On the opposite side of the stage, a 3D manipulator is placed with a small platinum wire loop on which a bead of pipette glass has been melted. (B) The unpolished pipette tip is brought into the same plane of view as the glass bead, but at opposite sides of the periphery. (C) When current is passed through the wire, the bead gets hot and expands into the center of the field. The tip of the pipette is transiently manipulated closer to the bead until the opening at the tip starts to close. (D) When the tip narrows and attains rounded edges, the pipette is withdrawn and the heat is turned oV. (E) A finished, polished pipette tip.
Table III Checklist for obtaining giga-ohm seals Cells are healthy CO2 regulation is accurate No trypsin for 24 h and no transfection reagents in past 12 h Maintained in salt solution at room temperature for less than 15 min Pipettes are functioning properly Pipettes are stored in dust-free box for 4 h after polishing Pipette solution filtered through 0.45 mm mesh Tip filled separately by capillary action before back filling; no bubbles Pipette holder gasket sized to glass and fits snugly (but do not over tighten) Suction line intact and dry No mechanical vibrations transmitted to tip (see Table II) Solutions Bath solution has more solutes than pipette solution Neither solution has proteins or ATP At least one of the solutions has 0.1 mM Ca2þ
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pipette after it is lowered in the bath, gentle positive pressure is applied on the suction line which is then pinched off until contact with the cell. In addition, bath solutions are designed to be 10% hyperosmotic to the pipette solution (usually by adding glucose), so water will stream out of the pipette tip. Axial approaches are more eVective than lowering the angled pipette straight down on to the cell. Some manipulators program a fourth axis or you can mount the manipulator at an angle so one axis is parallel to the pipette. We prefer to monitor the approach of the pipette to the cell surface by looking at the pipette current trace in response to a small voltage step. As the pipette touches the cell, the amplitude of the current diminishes. When it is reduced to 33–50%, we release the positive pressure on the pipette, and then, if necessary, we apply additional suction. To make these manipulations without disturbing the tip, the small tube connected to the pipette holder must be firmly anchored to the headstage and the microscope stage to prevent vibrations. Most of us prefer to apply the suction directly by mouth because the suction is controlled more easily and changes more evenly. Small flat cells are obviously harder to patch than large round ones. Visualization of the cell surface is improved by interference contrast optics, but this can be implemented on very simple microscopes for the price of two small pieces of black tape (Axelrod, 1981). Most people we have trained find the rat pituitary GH cell lines to be the easiest cells with which to learn patching. They can be obtained from the ATCC.
D. Making Recordings
1. Cell-Attached Patch Recordings The two most important things to remember about the cell-attached configuration are that the voltage polarities are reversed relative to traditional whole-cell recording and that there are two membranes in the current path between your electrodes. The amplifier sets the voltage between the pipette wire and the ground electrode in the bath, but, by convention, the cell interior is negative to the outside, so depolarizing the membrane of a cell-attached patch means making the pipette more negative. However, the cell membrane also contributes a voltage which is not clamped in the cell-attached configuration, so to accurately determine the voltage across the patch, the cell’s membrane potential must be set to zero by bathing the cell in an equimolar potassium solution of 140–150 mM. Unfortunately, that means all the voltage-gated channels, including the calcium channels, in the membrane outside the patch will be activated. Therefore, it is essential to reduce extracellular calcium to avoid flooding the cell with calcium. Some investigators use calcium buVers, but the bath is infinite relative to the cell volume and most cells are rapidly depleted of calcium. A more physiological solution is to reduce extracellular calcium to 0.1 mM, which is still 100 more than resting calcium inside the cell, but produces negligible currents in the presence of 2–5 mM magnesium. For cell-attached recordings, the primary goal is increasing the signal and reducing noise. Increasing the signal is usually achieved by increasing the
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concentration of the divalent ion in the pipette solution on the outside of the patch. Practically, 90 mM calcium is as high as one can achieve without making the pipette solution hyperosmotic to the bath. However, such large currents also invite unphysiological, or even toxic, calcium responses in the cytoplasm, so most people use 90 mM barium. The conductance of voltage-gated channels to barium is often higher than to calcium, and barium blocks many potassium channels, but high concentrations of divalent ions alter the surface potential of the membrane and shift voltage-activation and inactivation curves. If all the precautions that are described here are taken to reduce noise, it is possible to record unitary currents with physiological concentrations of calcium (Josephson et al., 2010). Patch pipettes with initial resistances greater than 10 MO rarely make high resistance seals, so several square micrometers of membrane are usually drawn into wider, lower resistance pipettes by the suction. Such large areas of membrane usually contain many diVerent types of channels, many of which have much larger unitary conductance than calcium channels. Therefore, sodium, potassium, and chloride must be replaced with impermeant ions and sometimes, ion channel blockers must be added too. Cesium does not permeate potassium selective channels, but it does go through many nonselective cation channels, so N-methyl-dglucamine (NMDG) is a safer substitute for sodium. Chloride can be replaced with methanesulfonic acid, although a few millimolar chloride must be left in to allow reversible current movement between the wire and the solution. To reduce background noise further, coating the pipette with Sylgard and obtaining higher resistance seals are the two most practical steps. Lowering the bath also helps although no perfusion system is perfect, so the bath cannot be too low. Stable recordings also require a drift-free manipulator or lifting the cell oV the bottom of the chamber, which is easier when calcium is removed from the bath solution. If you can lift the cell oV the substrate, the lowest noise recordings can be obtained by putting a layer of inert oil over the surface (Rae and Levis, 1992), but then the chamber must be designed with the perfusion inlet and outlet at the bottom.
2. Perforated Patch Recordings Many people report that they have tried perforated patch recording but could not get it to work. In our experience, there are two critical steps that befuddle most beginners until we show them the ‘‘secrets.’’ The two essential steps to successful perforated patch recording are eYcient solubilization of the antibiotic and optimal loading of the pipette (Horn and Marty, 1988). We learned how to sonicate from Robert Rosenberg, a calcium channel researcher at UNC Chapel Hill for many years, who also used bilayers. He taught us that cylindrical devices are most eVective, and they must be filled to the height where the water surface is most agitated. Then placing a covered, round bottom, cylindrical glass tube with less than 1 ml of solution into the vortex for less than a minute is suYcient to disperse the nystatin or amphotericin or gramicidin, but this only lasts for an hour or two.
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The second secret is empirically determining exactly how far to fill the tip with antibiotic-free solution. If the antibiotic is too close to the tip, it can permeabilize the entire cell membrane and prevents giga-ohm seal formation. If it is too far back, it takes forever for the perforation to proceed. Therefore, each person must take the time to figure out exactly how long to dip their pipettes in the antibioticfree solution by painstakingly examining and measuring how far up the solution goes for a given count; ‘‘one one thousand, two one thousand, etc.’’ With those preliminaries, we routinely get access resistances below 20 MO in 5 min and often it goes down closer to 10 MO. Finally, there is one mistake that even experienced electrophysiologists make that is much easier to avoid. Most patch clampers almost exclusively use the ‘‘whole cell’’ configuration, in which the membrane underneath the pipette is disrupted by suction, and the cell is dialyzed with the pipette solution. To achieve this configuration, they routinely add calcium chelators, such as EGTA, to the pipette solution. Chelating calcium destabilizes the patch membrane and prevents cytoplasmic calcium from rising to toxic levels. However, if EGTA is not removed for perforated patch recordings, suction often leads to cell dialysis with the antibiotic, and the current through its channels dwarfs the calcium current.
3. Recordings of Calcium Release-Activated Currents, Icrac The conductance of individual Icrac channels is too low to measure with the patch clamp technique although it has been estimated by fluctuation analysis (Prakriya and Lewis, 2006). While Icrac resulting from overexpression of STIM/ Orai in recombinant systems can be as large as 100 pA/pF at 100 mV, which translates into 1 nA whole-cell current in an average HEK293 cell, endogenous Icrac currents are only a few pA/pF or about 10 pA for the average cell. In order to measure whole-cell currents in the 1–10 pA/pF range, low-noise techniques that are usually only used for high-resolution single-channel current measurements need to be employed. Specifically, the whole-cell recording electrodes should be coated with an elastomer-like Sylgard 184 and, of course, the initial seal resistance prior to establishing whole-cell configuration should be as high as possible. With Sylgard-coated and subsequently fire polished electrodes, one should routinely obtain seal resistances of 50 GO in the cell-attached mode on HEK cells. At least for HEK293 cells, if calcium is buVered to 100 nM in the pipette, the wholecell configuration can then be obtained easily by a single, brief, and gentle suction pulse. In classical voltage jump protocols that are used to elicit whole-cell calcium currents, the eVect of carefully coating the pipette with Sylgard can be readily observed as a reduction of the fast capacitive transients at the beginning and end of the step. For Icrac measurements, however, investigators routinely use fast voltage-ramp protocols (typically 100 mV/s) to measure the quasi steady-state IV relationship of Icrac. Under those conditions, the contribution of capacitative current to the total current is less visible since it remains constant during the ramp,
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and small changes in the bath level that are almost inevitable during perfusion changes will change the amplitude of the capacitative current. This can produce a shift of the instantaneous current–voltage relationship along the current axis, which can easily be misinterpreted as a shift in the reversal potential of Icrac.
IV. Materials Although all patch clampers have their favorite vendors, as governmentemployed scientists, we cannot reveal our specific preferences here (see Table IV). If there is someone else at your institution who is patch clamping successfully, you should consult them first anyway and consider buying what they have after you try it because they will already be familiar with its operation.
V. Discussion The patch clamp methods described here are not easy. They have daunting initial costs in instrumentation, almost $100,000 at current list prices, and a steep learning curve for both the technical aspects and conceptual foundations of electrophysiology. Even for experienced investigators, the patch clamp technique is a laborious method that requires constant trouble shooting. Nevertheless, like any physical activity, patch clamp performance is always enhanced mysteriously by a belief in success. Thus, a common refrain around our laboratory is ‘‘If Neher or Sakmann could do it, then you can do it too.’’
Table IV Components of a patch clamp rig Tungsten wire for sylgarding and polishing Silver wire and pellets Pipette glass Pipette puller Dissecting microscope on swing arm for sylgard coating Pipette polisher with Inverted microscope with 40 LWD objective Coarse manipulator for adjusting heating element Fluorescent microscope with diVerential interference contrast optics LED illumination is cheaper, cooler, and electrically quieter 3D micromanipulator with submicron resolution and no drift Patch clamp amplifier, computer interface, and software Computer according to the software manufacturer’s specifications Physiological chamber and perfusion apparatus Pipette holder and agar bridge ground electrode Small cylindrical sonicator
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VI. Summary The patch clamp technique provides quantitative records of calcium channel activity in the plasma membrane at the molecular level in real time in situ. However, studying calcium channel function and regulation under physiological conditions in metabolically intact cells requires more demanding approaches than the conventional whole-cell recording through ruptured patches on dialyzed cells. Whole-cell recordings through antibiotic-perforated patches and single-channel recordings from cell-attached patches require additional eVort and attention to detail, but are currently unrivaled in their precision and detailed view of calcium channel function and physiological regulation of gating.
Acknowledgments Our studies have been supported by the NIH intramural program at NIEHS through Grant Z01ES080043. We also thank Sue Edelstein for drawing Figures 1 & 2.
References Aidley, D. J., and Stanfield, P. R. (1996). ‘‘Ion Channels: Molecules in Action.’’ 1st edn. Cambridge University Press, Cambridge, UK. Axelrod, D. (1981). Zero-cost modification of bright field microscopes for imaging phase gradient on cells: Schlieren optics. Cell Biophys. 3, 167–173. Conn, P. M. (1998). ‘‘Methods in Enzymology: Ion Channels Part B.’’ Vol. 293. Academic Press, New York, NY. Conn, P. M. (1999). ‘‘Methods in Enzymology: Ion Channels Part C.’’ Vol. 294. Academic Press, New York, NY. Erxleben, C., Liao, Y., Gentile, S., Chin, D., Gomez-Alegria, C., Mori, Y., Birnbaumer, L., and Armstrong, D. L. (2006). Cyclosporin and Timothy syndrome increase mode 2 gating of CaV1.2 calcium channels through aberrant phosphorylation of S6 helices. Proc. Natl. Acad. Sci. USA 103, 3932–3937. Hamill, O. P., Marty, A., Neher, E., Sakmann, B., and Sigworth, F. J. (1981). Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch. 391, 85–100. Hille, B. (2001). ‘‘Ion Channels of Excitable Membranes.’’ 3rd edn. Sinauer Associates, Inc., Sunderland, MA. Hogan, P. G., Lewis, R. S., and Rao, A. (2010). Molecular basis of calcium signaling in lymphocytes: STIM and ORAI. Annu. Rev. Immunol. 28, 491–533. Horn, R., and Marty, A. (1988). Muscarinic activation of ionic currents measured by a new whole-cell recording method. J. Gen. Physiol. 92, 145–159. Josephson, I. R., Guia, A., Lakatta, E. G., Lederer, W. J., and Stern, M. D. (2010). Ca(2þ)-dependent components of inactivation of unitary cardiac L-type Ca(2þ) channels. J. Physiol. 588, 213–223. Levis, R. A., and Rae, J. L. (1993). The use of quartz patch pipettes for low noise single channel recording. Biophys. J. 65, 1666–1677. Molleman, A. (2003). ‘‘Patch Clamping, An Introductory Guide to Patch Clamp Electrophysiology.’’ John Wiley & Sons, Chichester, England. Ogden, D. (1987). ‘‘Microelectrode Techniques: The Plymouth Workshop.’’ 2nd edn. The Company of Biologists Limited, Cambridge, UK.
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Prakriya, M., and Lewis, R. S. (2006). Regulation of CRAC channel activity by recruitment of silent channels to a high open-probability gating mode. J. Gen. Physiol. 128, 373–386. Rae, J. L., and Levis, R. A. (1992). A method for exceptionally low noise single channel recordings. Pflugers Arch. 420, 618–620. Rudy, R., and Iverson, L. E. (1992). ‘‘Methods in Enzymology: Ion Channels.’’ Vol. 207. Academic Press, New York, NY. Sakmann, B., and Neher, E. (1995). ‘‘Single-Channel Recording.’’ 2nd edn. Plenum Press, New York, NY.
CHAPTER 8
Nuclear Patch-Clamp Recording from Inositol 1,4,5-Trisphosphate Receptors Taufiq Rahman and Colin W. Taylor Department of Pharmacology Tennis Court Road, University of Cambridge Cambridge, United Kingdom
Abstract Introduction Nuclear Patch-Clamp Recording Choice of Cells for Analyses of IP3R Methods A. Culture of DT40 Cells B. Isolation of Nuclei C. Solutions for Patch-Clamp Recording D. Patch-Clamp Recording E. Analysis of Single-Channel Records V. Concluding Remarks References
I. II. III. IV.
Abstract Inositol 1,4,5-trisphosphate receptors (IP3R) are ubiquitous intracellular Ca2þ channels. They are regulated by IP3 and Ca2þ and can thereby both initiate local Ca2þ release events and regeneratively propagate Ca2þ signals evoked by receptors that stimulate IP3 formation. Local signaling by small numbers of IP3R underpins the utility of IP3-evoked Ca2þ signals as a ubiquitous signaling pathway. The physiological impact of Ca2þ release by very small numbers of IP3R underscores the necessity to understand the behavior of IP3R at the single-channel level. In addition, and in common with analyses of every other ion channel, singlechannel analyses have the potential to define the steps linking IP3 binding to channel opening. Patch-clamp recording, by resolving the openings and closings of single channels with exquisite temporal resolution, is the most powerful METHODS IN CELL BIOLOGY, VOL. 99 Copyright 2010, Elsevier Inc. All rights reserved.
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technique for analysis of single-channel events. It has contributed enormously to the understanding of gating and desensitization/inactivation of numerous ion channels. However, most IP3R reside within intracellular membranes, where they are inaccessible to conventional patch-clamp recording methods. Here, we describe the application of nuclear patch-clamp methods to single-channel analyses of native and recombinant IP3R.
I. Introduction Inositol 1,4,5-trisphosphate receptors (IP3R) comprise a family of tetrameric intracellular channels that mediate the release of Ca2þ from the intracellular stores of almost all animal cells (Foskett et al., 2007; Taylor et al., 1999). Three genes encode homologous subunits of vertebrate IP3R, and a single gene encodes invertebrate IP3R. The key structural determinants of IP3R activation, although presently poorly understood, are likely to be similar for all IP3R. Activation is initiated by binding of IP3 to a conserved IP3-binding core toward the N-terminal of each subunit (Bosanac et al., 2002), conformational changes then pass via the N-terminal suppressor domain (Bosanac et al., 2005) to the pore, which is formed by transmembrane regions lying toward the C-terminus (Boehning and Joseph, 2000; Foskett et al., 2007; Rossi et al., 2009; Taylor et al., 2004). Most IP3R in most cells are expressed within membranes of the endoplasmic reticulum (ER). DiVerent IP3R subtypes may, however, diVer in their subcellular distributions (Taylor et al., 1999) and in their modulation by various additional signals and associated proteins (Betzenhauser et al., 2008b; Choe and Ehrlich, 2006; Mackrill et al., 1997; Patterson et al., 2004; Wojcikiewicz and Luo, 1998). Resolving the roles of diVerent IP3R subtypes in the genesis of the complex Ca2þ signals that regulate cellular activity is an important issue (Futatsugi et al., 2005; Miyakawa et al., 1999; Sugawara et al., 1997; Wang et al., 2001). Opening of the intrinsic pore of all IP3R requires binding of IP3 and Ca2þ (Adkins and Taylor, 1999; Marchant and Taylor, 1997). IP3R can, therefore, both initiate the Ca2þ signals evoked by receptors that stimulate IP3 formation and then regeneratively propagate them by Ca2þ-induced Ca2þ release. This dual regulation of IP3R allows a hierarchical recruitment of Ca2þ release events as the stimulus intensity increases (Bootman et al., 1997; Marchant and Parker, 2001). Single IP3R respond first, then several IP3R within a cluster open together to give larger local events (‘‘puVs’’), and as puVs become more frequent, they ignite regenerative Ca2þ waves (Bootman and Berridge, 1995; Marchant et al., 1999). This hierarchy of events allows Ca2þ to function as a local or global messenger, a feature that underlies its versatility (Berridge et al., 2000). A key point for the present discussion is that local events involving very few IP3R underlie the Ca2þ signals that regulate cellular activity. By contrast, for most ion channels, it is the collective behavior of large numbers of channels, the macroscopic current, that determines the physiological response, a change in membrane potential, or
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transcellular ion flux, for example. The distinction highlights the particular importance of single-channel recording in the analysis of IP3R. As with all such analyses, they provide the highest resolution insight into the opening and closing of single channels and can thereby reveal details of gating mechanisms (Colquhoun, 2007; Sivilotti, 2010). But for IP3R and other Ca2þ channels too, openings of individual channels are the physiologically significant behavior. The patch-clamp technique, developed originally by Neher and Sakmann (1976) with subsequent improvements (Hamill et al., 1981), is the most powerful means of studying the behavior of ion channels in their native environment. It involves recording currents passing through an electrically isolated, small area (‘‘patch’’) of biological membrane in response to an applied voltage or ionic gradient (Fig. 1A and B). Isolation of the patch is achieved by pressing a polished glass pipette tip of 1 mm diameter (containing electrolyte solution) against the cellsurface and applying gentle suction to form a very high-resistance ‘‘giga-Ohm’’ (GO) seal (Hamill et al., 1981). The tight seal is crucial because it isolates the patch both electrically and physically, so reducing background noise and allowing singlechannel events to be resolved (Hamill et al., 1981). Because of the electrical isolation and low resistance of the patch-pipette relative to the membrane, a patch can be voltage-clamped by simply applying a potential to the pipette. These patch-clamp recordings allow the openings and closings of individual channels to be resolved with submillisecond temporal resolution under optimal conditions (Fig. 1C). The amplitudes of these tiny currents and their dependence on applied potential and ion concentrations allow the ion selectivity and rates of ion permeation to be determined. Lurking within the pattern of stochastic openings and closings is the information from which the sequence of events that leads to channel gating and desensitization/inactivation can be reconstructed. Comprehensive descriptions of the patch-clamp technique are available from the original articles (Hamill et al., 1981; Neher and Sakmann, 1976) and subsequent reviews (Ogden, 1994; Sakmann and Neher, 1995). However, most IP3R are expressed in membranes of the ER, where they are inaccessible to conventional patch-clamp techniques. Alternative approaches are therefore needed.
II. Nuclear Patch-Clamp Recording It is impracticable, despite one heroic success recording from IP3R within the ER of an intact cell (Jonas et al., 1997), to use patch-clamp techniques routinely to record the behavior of single channels within the membranes of intracellular organelles in situ. A more promising approach for single-channel recordings in situ is the ‘‘optical patch-clamp,’’ where high-resolution optical microscopy in combination with fluorescent Ca2þ indicators is used to measure the Ca2þ signals evoked by opening of single or small clusters of IP3R (Demuro and Parker, 2007; Smith and Parker, 2009). Presently, however, these optical methods can be used only to measure fluxes through Ca2þ channels, and they lack the temporal
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A
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Reference electrode
Bath B
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Fig. 1 Conventional patch-clamp recording. (A) A polished glass pipette forms a tight seal against a biological membrane isolating an area across which the tiny currents passing through small numbers of open channels can be recorded. (B) DiVerent configurations of conventional patch-clamp recording. Beginning with a cell-attached patch, an inside-out excised patch can be produced by pulling the patchpipette away from the cell, while suction or a strong brief voltage-pulse ruptures the underlying membrane to give the whole-cell configuration. An outside-out patch can then be produced by pulling the patch-pipette away from the whole-cell configuration. (C) Typical whole-cell recordings of IP3R3 expressed in the plasma membrane of DT40-KO cells expressing rat IP3R3. PS included IP3 (10 mM), ATP (5 mM), and a free [Ca2þ] of 200 nM; Kþ was the charge carrier and the holding potential was 100 mV. C, O1, and O2 show the closed state and the openings of 1 and 2 IP3R, respectively.
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resolution of conventional patch-clamp recording. Measuring the electrical activity of intracellular channels, therefore, presently relies upon redirecting channels to the plasma membrane, where they then become accessible to conventional patch-clamp techniques (Xu et al., 2007); reconstituting the channel into an artificial membrane; or isolating organelles that express the channel and adapting the patch-clamp technique to record from these membranes. The latter has been used, for example, to resolve the behavior of the mitochondrial Ca2þ uniporter from mitochondria stripped of their outer membrane (‘‘mitoplasts’’) (Kirichok et al., 2004) and for single-channel recordings of the endolysosomal protein, TRPML1, from artificially enlarged lysosomes (Dong et al., 2008). All three methods have been used to record single-channel behavior of IP3R. We observed that DT40 cells express very small numbers of functional IP3R within the plasma membrane (Dellis et al., 2006) and because DT40 cells lacking native IP3R are available (Section III), conventional whole-cell patchclamp recording has been used by us (Dellis et al., 2008) and others (Betzenhauser et al., 2008b, 2009a) to examine the behavior of recombinant and mutant IP3R. Typical recordings from IP3R in the plasma membrane of DT40 cells are shown in Fig. 1C. A limitation of this approach is that excised patch-clamp recording, where the ‘‘intracellular’’ composition can be precisely controlled, is impracticable (because plasma membrane IP3R are too scarce), and with wholecell recording, it is diYcult to define reliably the exact concentration of IP3 bathing the IP3R (Dellis et al., 2006). Detailed descriptions of the methods used for wholecell recording of IP3R expressed in the plasma membrane of DT40 cells have been published (Dellis et al., 2006; Taylor et al., 2009b). We prefer nuclear patchclamping (see below) to whole-cell recording because the nuclear envelope is continuous with the ER (Fig. 2A), wherein reside most IP3R, and it is practicable to work with excised patches that provide better control of media bathing both sides of the membrane. The first electrical recordings from IP3R were made by incorporating native or purified IP3R into artificial lipid bilayers (Bezprozvanny et al., 1991; Ehrlich and Watras, 1988; Maeda et al., 1991; Mayrleitner et al., 1991). As with all reconstituted systems, the lipid composition of the bilayer and the steps involved in isolating, purifying, and reconstituting IP3R into the bilayer may aVect normal function of the channel, not least its regulation by accessory proteins (Boehning et al., 2001a; Foskett et al., 2007; Patterson et al., 2004). Anecdotally, and it equates with our experience, it seems to be more diYcult to obtain bilayer recordings from IP3R than from its close relatives, the ryanodine receptors (Williams, 1995). Many of the problems with bilayer recording are resolved by using nuclear patchclamp recording (Fig. 2). This technique was first introduced in the early 1990s (Matzke et al., 1990; Mazzanti et al., 1990, 2001; Tabares et al., 1991) and subsequently, applied by the laboratories of Clapham (Stehno-Bittel et al., 1995) and Foskett (Mak and Foskett, 1994) to record single-channel events from native IP3R in nuclei from Xenopus oocytes. It has, subsequently, been successfully applied
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A
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Fig. 2 Nuclear patch-clamp recording of IP3R. (A) The nuclear envelope comprises an inner (INM) and outer (ONM) membrane surrounding a luminal space that is continuous with the lumen of the ER. The continuity of the ONM with the ER membrane allows some ER proteins to invade the ONM, where they become accessible to nuclear patch-clamp recording. (B) Phase-contrast image of a DT40 nuclear preparation showing nuclei, one of which has debris attached, and an intact cell. (C) Three recording configurations are used for nuclear patch-clamp recording. The on-nucleus, lumen-out, and cytosol-out excised patch configurations are analogous to the cell-attached, inside-out, and outside-out configurations of conventional patch-clamp recording (Fig. 1B). (D) An excised lumen-out nuclear patch illustrating the convention used to report membrane potential. (E) Typical recording from a single IP3R3 recorded in the lumen-out configuration from the nucleus of a DT40-KO cell stably expressing rat IP3R3. PS included IP3 (10 mM), ATP (5 mM), and a free [Ca2þ] of 200 nM; Kþ was the charge carrier and the holding potential was þ40 mV. C denotes the closed state.
to analyses of diVerent IP3R subtypes expressed in diVerent cells, including COS-7 cells (Boehning et al., 2001a), insect Sf9 cells (Ionescu et al., 2006), smooth muscle (Kusnier et al., 2006), DT40 cells (Betzenhauser et al., 2009b; Dellis et al., 2006;
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Rahman et al., 2009), human B lymphoblasts (Cheung et al., 2008), cerebellar Purkinje cells (Marchenko et al., 2005), and embryonic cortical neurons and fibroblasts (Cheung et al., 2008). The utility of nuclear patch-clamp recording derives from the fact that the outer nuclear envelope is continuous with the ER membrane (Dingwall and Laskey, 1992) (Fig. 2A). Channels that are normally expressed within ER membranes can, therefore, pass into the outer nuclear envelope, allowing their activity to be recorded from patches of nuclear membrane in a near-physiological setting (Fig. 2). Abundant nuclear pore complexes, each with a large central conduit linking cytoplasm and nucleoplasm (Mazzanti et al., 2001), might have been expected to compromise formation of the tight seals required for patch-clamp recording or at least pollute recordings from conventional channels with lesser conductances. In practice nuclear pores appear not to cause problems. It seems unlikely, though it remains possible, that this results from patching onto ER overlying the nuclear envelope, rather than the envelope itself. It is perhaps more likely that patches that include nuclear pores are rejected because they fail to form giga-Ohm seals, or the high-Kþ medium used for nuclear patch-clamp recording favors closure of nuclear pores (Bustamante and Varanda, 1998).
III. Choice of Cells for Analyses of IP3R Almost all animal cells express IP3R, most express more than one of the three vertebrate gene products, and substantial alternative splicing and posttranslational modifications add further to the diversity of subunits from which IP3R are assembled (Foskett et al., 2007). Assembly of these subunits into homo- and heterotetrameric structures increases the diversity of functional IP3R enormously (Joseph et al., 1995, 2000; Wojcikiewicz and He, 1995). Despite this complexity, there have been many valuable studies of the single-channel behavior of native IP3R in, for example, Xenopus oocytes, nuclei from oocytes, insect Sf9 cells, and cerebellar Purkinje neurons, and of native IP3R reconstituted into lipid bilayers (Section II). But the limitations of such studies are obvious when it to comes to exploring the structural basis of IP3R activation. This demands a more homogenous population of IP3R with a defined structure and ideally expressed in a native membrane. At present, only one expression system provides the ‘‘null background’’ that allows these demanding criteria to be satisfied: DT40 cells (Fig. 3). DT40 cells originate from an avian leukosis virus-transformed bursal B cell (Baba et al., 1985). The uniquely valuable feature of these cells is the unusually high frequency with which they integrate targeted DNA constructs into their genome (Buerstedde and Takeda, 1991). This feature, together with the shorter introns of avian genes, allows targeted disruption of specific genes and has ensured widespread use of DT40 cells for ‘‘gene-knockouts.’’ In a monumental eVort, Kurosaki and his colleagues used targeted gene disruption to inactivate both copies of all three IP3R genes in DT40 cells and thereby to generate the first cell
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Fig. 3 DT40-KO cells provide a null background for expression of functional IP3R. (A) IP3-evoked Ca2þ release from permeabilized DT40 cells assessed using a luminal Ca2þ indicator (Tovey et al., 2006). Permeabilized DT40-KO cells stably expressing rat IP3R3 (DT40-R3) release Ca2þ when stimulated with IP3, whereas DT40-KO cells are unresponsive. (B) Currents recorded from lumen-out patches from DT40-KO and DT40-R3 cells. PS included IP3 (10 mM), ATP (5 mM), and a free [Ca2þ] of 200 nM; Kþ was the charge carrier and the holding potential was þ40 mV. C denotes the closed state.
line lacking functional IP3R (Sugawara et al., 1997). These DT40-KO cells, which Kurosaki (RIKEN, Japan) has made widely available, provide the only null background for functional expression of IP3R (Fig. 3). They have been extensively used by many groups to express each of the three IP3R subtypes and define their functional properties, and to explore the role of IP3R in many higher order processes (e.g., Joseph and Hajnoczky, 2007; Miyakawa et al., 1999). A recent review provides further details of the use of DT40 cells for analyses of Ca2þ signaling pathways (Taylor et al., 2009b). Here, we describe our use of DT40 cells expressing mammalian IP3R for nuclear patch-clamp recording.
IV. Methods A. Culture of DT40 Cells Wild-type DT40 cells (Cell Bank number RCB1464) and DT40-KO cells (RCB1467) are available from Riken Bioresource Center Cell Bank, Japan (http://www.brc.riken.jp/lab/cell/). Cells are grown in RPMI 1640 medium (Invitrogen) supplemented with 10% fetal bovine serum (FBS, Sigma), 2 mM l-glutamine, 1% chicken serum (Sigma), and 50 mM b-mercaptoethanol (Invitrogen) in a humidified atmosphere containing 5% CO2, ideally at 39–41 C (matching the increased body temperature of birds). It is, however, acceptable and more convenient when incubators are shared with mammalian cells to culture DT40 cells at 37 C without loss of viability. The only obvious eVect is a slowing of growth rate; the doubling time has been reported to increase from about 10 h at 39–41 C to 18 h at 37 C (Mak et al., 2006). The chicken serum must be heat-inactivated
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(56 C for 30 min). We avoid antibiotics, but addition of penicillin (10,000 units/ ml) and streptomycin (10 mg/ml) to culture media is optional (Winding and Berchtold, 2001). The cells grow in suspension in flasks, Petri dishes, or multiwell plates (Greiner Bio-one). Cells are passaged by 20-fold dilution every 2–3 days when they reach a density of about 2 106 cells/ml. Avoid growing cells beyond a density of 2.5 106 cells/ml. Cells (2 106ml 1) in either culture medium or FBS supplemented with 10% dimethylsulfoxide (DMSO, Sigma) can be frozen and then stored in liquid nitrogen following standard procedures. We routinely culture cells for 30–35 passages, before thawing a new frozen stock. For the latter, 1 ml of cells is dispensed into 20 ml of medium. We find it unnecessary to remove residual DMSO at this stage. Cells are then passaged after 24 h. DT40 cells are not easy to transfect with IP3R expression constructs, we therefore use cell lines stably expressing rat IP3R1 (GenBank accession number of GQ233032.1), mouse IP3R2 (AB182290), and rat IP3R3 (GQ233031.1). Details of the methods and sources of the original clones are provided in previous publications (Dellis et al., 2006; Rahman et al., 2009; Rossi et al., 2009; Tovey et al., 2010). Briefly, DT40-KO cells are transfected by nucleofection with linearized constructs of pcDNA3.2-IP3R using solution T and program B23 (Amaxa) using 5 mg DNA/106 cells. G418 (Invitrogen, 2 mg/ml) is used for selection. Expression of IP3R in each cell line is quantified by immunoblotting using custom-made antipeptide antisera (Cardy et al., 1997; Dellis et al., 2006; Rossi et al., 2009; Tovey 3 et al., 2010) and, where needed, by H-IP3 binding. Functional expression of IP3R in each DT40 cell line is verified by comparison with DT40-KO cells using a luminal Ca2þ indicator and a high-throughput assay for IP3-evoked Ca2þ release (Laude et al., 2005; Tovey et al., 2006) (Fig. 3). Only cell lines shown to express functional IP3R are used for nuclear patch-clamp recording. B. Isolation of Nuclei Several methods have been described for isolation of nuclei; most rely on a combination of osmotic and mechanical lysis of cells (Boehning et al., 2001a; Bustamante, 1994; Franco-Obregon et al., 2000; Marchenko et al., 2005). Our protocol is adapted from that of Boehning et al. (2001a). DT40 cells expressing a recombinant IP3R (DT40-IP3R cells, 1.5–2 106 cells/ml) are centrifuged (500 g for 2 min at 4 C), washed once with ice-cold phosphate-buVered saline (PBS), and then once with cold nuclear isolation medium (NIM). PBS has the following composition: 137 mM NaCl, 2.7 mM KCl, 10 mM Na2PO4, KH2PO4, pH 7.4, with NaOH. NIM comprises: 250 mM sucrose, 150 mM KCl, 3 mM b-mercaptoethanol, 10 mM Tris–HCl, 1 mM phenylmethanesulfonyl fluoride (PMSF, Sigma), pH 7.5. Cell pellets are resuspended in NIM supplemented with complete protease inhibitor cocktail (Roche, 1 mini-tablet/20 ml) and stored on ice for up to 4–5 h. For isolation of nuclei, 1 ml of the cell suspension is homogenized with 3–4 strokes of a Dounce homogenizer (Wheaton Industries, Inc.), which lyses about 5–10% of cells, assessed by staining with Trypan Blue (0.001%). This crude lysate
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containing some isolated nuclei is stored in NIM on ice and used within 1 h for patch-clamp experiments. Because the activity of nuclear IP3R has been reported to decrease after 40 min at 20 C (Boehning et al., 2001a), we routinely prepare fresh nuclei at hourly intervals. DT40 cells are not much larger than their nuclei (Fig. 2B). The inexperienced eye may therefore find it diYcult to distinguish nuclei from cells. But nuclei rarely have the smooth surface of intact cells and they often only partially protrude from broken cells, where the relatively clean exposed surface allows formation of a giga-Ohm seal. The yield of nuclei can be substantially increased to 50–60% using methods that require incubation in hypo-osmolar media (Franco-Obregon et al., 2000), but we rarely detect active IP3R after such isolation procedures. A nuclear isolation kit (Sigma Nuclei EZ Prep) also provides nuclei in high yield ( 85%), but we rarely succeed in forming giga-Ohm seals with these nuclei. In practice, the low yield of nuclei with our protocol is not a limitation for nuclear patch-clamp recording. IP3R have also been reported to be expressed within the inner nuclear membrane (Humbert et al., 1996; Marchenko et al., 2005). The citrate treatment used to remove the outer nuclear membrane and so allow patch-clamp recording of IP3R within the inner membrane (Marchenko et al., 2005) appears not, at least in our experience, to be readily applicable to DT40 cell nuclei.
C. Solutions for Patch-Clamp Recording For most recordings, we use Kþ as the charge-carrier. This eliminates the complexity of having Ca2þ passing through the IP3R regulate its activity, and it provides larger single-channel currents than with bivalent cations (Rahman et al., 2009). The bath solution (BS), which bathes the luminal surface of the nuclear envelope, typically contains 140 mM KCl, 10 mM HEPES, 100 mM 1,2-bis(2aminophenoxy)ethane-N,N,N0 ,N0 -tetraacetic acid (BAPTA, tetra potassium salt, Calbiochem), and a free [Ca2þ] of 200 nM (total CaCl2, 51 mM) adjusted to pH 7.1 with KOH. The usual pipette solution (PS), which bathes the cytosolic surface of the membrane, contains 140 mM KCl, 10 mM HEPES, 500 mM BAPTA, Na2ATP (0.5 mM), IP3 (American Radiolabeled Chemicals, Inc.), and a free [Ca2þ] of 200 nM (total CaCl2, 254 mM) adjusted to pH 7.1 with KOH. IP3, Ca2þ, and ATP are the three ligands of IP3R whose concentrations must be adjusted to obtain optimal IP3R activity in patch-clamp recording (Foskett et al., 2007). The concentration of IP3 in PS can be varied between experiments, depending on the aim of the analysis; 10 mM will usually be suYcient to saturate responses to IP3 (Foskett et al., 2007; Rahman et al., 2009). The potentiating eVects of ATP diVer between IP3R subtypes with higher concentrations required optimally to activate IP3R3 (Betzenhauser et al., 2008a; Miyakawa et al., 1999). The pH of PS must be readjusted after addition of ATP, and its eVects on free [Ca2þ] also need to be considered. Finally, because Mg2þ aVects the conductance of IP3R (Mak and Foskett, 1998; Rahman and Taylor, 2009), it is advisable to use ATP of the highest
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purity. Freshly prepared dilutions of ATP and IP3 (from frozen stocks) are added to PS as required. EVective buVering of the free [Ca2þ], which might reasonably be varied between nanomolar and several micromolar, requires buVers with appropriate aYnities for Ca2þ (Patton et al., 2004). For free [Ca2þ] less than 1 mM, BAPTA (KDCa 192 nM at pH 7.4) is preferable to EGTA because it has faster Ca2þbinding kinetics and lesser pH-dependence. Where the free [Ca2þ] of PS is 1–100 mM, we use 5,50 -dibromo BAPTA (KDCa 1.83 mM, Fluka), EGTA (KDCa 67 nM), and/or N-(2-hydroxyethyl)ethylenediamine-N,N0 ,N0 -triacetic acid (HEDTA, KDCa 2.2 mM, Sigma), alone or in appropriate combinations (Bers et al., 1994). We initially estimate the amount of CaCl2 required to achieve the desired free [Ca2þ] using WinMaxC software (http://www.stanford.edu/cpatton/maxc.html) and then measure the free [Ca2þ] of the final media (supplemented with ATP, IP3, etc.) directly using either a fluorescent Ca2þ indicator (Fluo-3, KDCa ¼ 325 nM, Invitrogen) or a Ca2þ-sensitive electrode (Mettler Toledo Ingold, Fisher Scientific) for higher free [Ca2þ] (Dellis et al., 2006; Rahman et al., 2009). The osmolarities of all solutions are adjusted to 290–310 mOsm kg 1 using glucose and mannitol, and verified using a vapor pressure osmometer (Wescor, Inc.). This is more important for recordings in the on-nucleus configuration than for recordings from excised patches (Fig. 2C). PS is prepared to be slightly ( 10%) hypo-osmolar to BS to aid formation of giga-Ohm seals (Hamill et al., 1981). All recording solutions are filtered using detergent-free 0.2-mm filters (AcrodiscÒ syringe filters, Pall Corporation) (Ogden, 1994). Fresh recording solutions (without added IP3 or ATP) are prepared monthly and stored at 4 C. The presence within the nuclear envelope of other large-conductance cation and Cl channels (Franco-Obregon et al., 2000; Marchenko et al., 2005; Mazzanti et al., 2001; Tabares et al., 1991) might potentially contaminate recordings of nuclear IP3R. In practice, this appears not to be a significant problem. If such problems should arise, they can be mitigated by replacing KCl in BS and PS with cesium methanesulfonate (CsCH3SO3): Csþ permeates IP3R but not Kþ channels (Tovey et al., 2010), while most anion channels are impermeable to CH3SO3. D. Patch-Clamp Recording The equipment required for nuclear patch-clamp recording is the same as that used for conventional patch-clamp recording (Fig. 1A). The basic rig includes an amplifier, headstage, electrode holder, micromanipulator, AgCl bath electrode, data acquisition system (i.e., analog-to-digital converter, computer, and software), inverted microscope, air table, and a Faraday cage. In addition, a pipette puller and fire-polisher or microforge are required to fabricate electrodes. Optional extras include systems for exchange of solutions, an oscilloscope, and a low-pass 8-pole Bessel filter; the latter extends the filtering range down to 0.1 Hz from the 1 to 100 kHz provided by the inbuilt filter. Comprehensive descriptions of the
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equipment used for patch-clamp recording are presented in relevant chapters of Sakmann and Neher (1995). Among the many steps taken to minimize electrical noise, the following are particularly important: appropriate grounding of equipment, use of thick-walled glass capillaries, filling pipettes with PS to the minimal level required to contact the recording electrode, and minimizing immersion of the pipette in BS (Rae and Levis, 1992). Pipettes are pulled from filamented, thick-walled borosilicate glass capillaries (GC150-10F, Clark Electromedical Instruments) using a Flaming/Brown P-87 horizontal micropipette puller (Sutter Instruments), and then fire-polished to a tip diameter of 1 mm using a microforge (MF-830, Narishige). It is advisable to melt a small bead of glass onto the wire of the forge to prevent platinum vapor from reaching the pipette tip. With monovalent cations as charge carriers, the single-channel conductance (g) of IP3R is large enough ( 360 pS, Section IV.E) to achieve good signal-to-noise ratios without hydrophobic coating of the patchpipette (Penner, 1995). But when g of IP3R is reduced, with pore mutants or with Ca2þ or Ba2þ as charge carriers, for example, it may be necessary to coat pipette TM tips with Sylgard (Dellis et al., 2006, 2008). When filled with PS, the pipette resistance typically remains within the range of 15–20 MO. Pipettes are best prepared a few hours before experiments. Unused pipettes can, however, be stored in an air-tight container and used later, but it is advisable to repolish them lightly before use to remove any impurities accumulated during storage. Petri dishes are precoated with poly-l-ornithine or poly-l-lysine (0.01%, Sigma) for 1–2 h, then rinsed twice with deionized water and air-dried. The nuclear preparation (15 ml) is added to a Petri dish containing BS (1.5 ml) and the cells/ nuclei are allowed to adhere. The dish is then mounted on the stage of an inverted microscope (Zeiss Axiovert 100) coupled to an assembly of headstage (CV 203 BU, Molecular Devices) and micromanipulator (PCS-1000, Burleigh Instruments). Recordings are made at room temperature ( 20 C) in the on-nucleus or excised configuration (Fig. 2C). The latter is preferable because it allows control of the medium on both sides of the membrane and eVective control of the voltage across the patch. A nucleus largely free from debris is first identified (Fig. 2B) and the patchpipette is positioned, using the micromanipulator, with its tip just above the nucleus. A slight positive pressure is applied to the inside of the patch-pipette before dipping it into the BS to avoid dirt accumulating at the pipette tip and to prevent backflow of BS into the PS (Hamill et al., 1981). After dipping the pipette into BS, the pipette capacitance is compensated using the specific oVset on the amplifier and the pipette resistance (typically 10–15 MO) is noted. As the pipette tip approaches the nucleus, the positive pressure is relieved. Taking care not to puncture the nuclear membrane, the pipette is lowered until it contacts the membrane, which should increase the pipette resistance by at least 2 MO. A giga-Ohm seal ( 5 GO) usually forms within a few seconds of applying slight negative pressure, by suction, to the inside of the pipette; this is usually controlled by an attached 50-ml syringe or by mouth. Seal formation can sometimes be facilitated
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by applying a holding potential of about 40 mV once a high-resistance contact is established (Ogden, 1994), and occasionally giga-Ohm seals form spontaneously. Seals > 5 GO can be routinely obtained with mild suction providing the nucleus is immobile and free of debris. To form an excised patch, our preferred recording configuration, the patch is pulled from the nucleus after forming the giga-Ohm seal (Fig. 2C). To prevent formation of closed vesicles at the tip of the patch-pipette, excised patches are briefly (1–2 s) exposed to air and then reimmersed in BS (Hamill et al., 1981). Prewritten protocols are then used to record currents through the excised patch at diVerent holding potentials. The bath electrode is grounded (i.e., 0 mV) and for convenience, the potential across a nuclear patch (whether attached or excised) is defined as the pipette potential minus the bath potential. That is, with symmetrical media, a positive holding potential would favor movement of cations from PS (the cytosolic surface) into BS (the luminal surface) producing an outward current and an upward deflection on the channel record (Fig. 2D and E) (Franco-Obregon et al., 2000; Mak and Foskett, 1994; Rahman et al., 2009). For determination of current–voltage (I–V) relationships, and thereby the single-channel conductance (g) of the channel (Section IV.E), the voltage across the excised patch can be stepped from 60 to þ 60 mV in increments of 20 mV from a holding potential of 0 mV. Applying more extreme voltages aVects the stability of the nuclear patch. For all other experiments, including kinetic analyses, currents are typically recorded at þ 40 mV for between 1 and 10 min. Currents are amplified with an Axopatch 200B amplifier in its voltage-clamp mode, filtered at 1 kHz with a low-pass 4-pole Bessel filter (built into the amplifier), and digitized at 10 kHz with a Digidata 1322A interface using the PC-based acquisition software package pClamp 9.2 (Molecular Devices) (Colquhoun, 1994). This filtering, while it inevitably causes some loss of information, has the eVect of rejecting signals (background noise) that are too brief to reflect the gating of IP3R. If the filtering frequency is set too low, it will reject events that do reflect gating of channels, and if set too high, background noise will obscure the openings. The optimal filtering frequency is, therefore, a compromise that depends upon the noise and time-course of the channel events; it needs to be optimized empirically. The sampling rate must, of course, exceed the filter frequency if further valuable information is not to be lost as the signals are digitized. In practice, digitization should be 10–20 times faster than the cutoV or ‘‘corner’’ frequency of the filter (Colquhoun, 1994). Most nuclear patch-clamp studies of IP3R have used 1-kHz filtering (Dellis et al., 2006; Ionescu et al., 2006; Mak and Foskett, 1997; Rahman et al., 2009). For presentation, traces can be further filtered oZine using a Gaussian filter (built within ClampFit). For the determination of relative permeabilities to cations, asymmetric recording solutions are used. For example, the normal BS can be replaced by a Ba2þ-rich BS (50 mM BaCl2, 30 mM KCl, 10 mM HEPES, adjusted to pH 7.1 with KOH), while the PS remains unchanged (Boehning et al., 2001a; Dellis et al., 2006). The liquid junction potential (LJP) under this asymmetric condition can be predicted (5.2 mV at 20 C), using the ‘‘junction potential calculator’’ (JPCalc, within
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pClamp 9.2), which uses the generalized Henderson equation (Barry, 1994). The calculated LJP is then subtracted from the observed reversal potential (Erev) obtained from the current–voltage (I–V) plot. We return in Section IV.E, after considering analysis of the raw traces, to describe how Erev allows the relative permeability of IP3R to diVerent cations to be calculated. In both the on-nucleus and excised patch configurations described above, the cytoplasmic surface of the IP3R lies within the patch-pipette (Fig. 2C); it is, therefore, diYcult to change the IP3 concentration once the giga-Ohm seal has formed. It is possible, though diYcult, to perfuse a patch-pipette and thereby to vary the composition of the ‘‘cytosolic’’ medium while recording channel activity (Hering et al., 1987; Maathuis et al., 1997), but this technique has not yet been applied to IP3R. Other options include the cytoplasm-out configuration of nuclear patch-clamp recording (Fig. 2C), which has been successfully applied to analyses of IP3R in Sf9 cells (Mak et al., 2007). Alternatively, flash-photolysis of caged-IP3 within the patch-pipette in either the on-nucleus or excised nuclear patch configuration can be used rapidly to increase the IP3 concentration bathing the cytosolic surface of the IP3R once the recording is underway (Rahman et al., 2009). For these flash-photolysis experiments, pipettes are prepared from thin-walled, nonfilamented borosilicate glass capillaries (Harvard Instruments) and PS includes d-myo-inositol 1,4,5-trisphosphate, (4,5)-1(2-nitrophenyl) ethyl ester (caged-IP3, 100 mM, Calbiochem). After recording for 30–60 s, IP3 can then be released into PS by photolysis of caged-IP3 using a single highintensity flash (1 ms) from a Xe-flash lamp (XF-10, Hi-Tech Scientific; 240 J with the capacitor charged to 385 V) passed through a filter (300–350 nM) (Walker et al., 1987). A problem with this approach is the diYculty in assessing the concentration of IP3 to which the IP3R are exposed after flash-photolysis of caged-IP3. E. Analysis of Single-Channel Records Two sorts of information can be extracted from single-channel records: the properties of the open channel (its ability to conduct diVerent ions); and the sequence of stable states through which the channel passes as it moves between closed, open, and desensitized conditions. Here, we provide only a brief introductory summary of the methods used to extract this information from the openings and closings of channels resolved by patch-clamp recording. The reader interested in more rigorous and detailed descriptions is advised to begin with two excellent books (Ogden, 1994; Sakmann and Neher, 1995). Several software packages are available for analysis of electrophysiological records. These include ClampFit, which includes the pClamp suite (Molecular Devices), and DC-soft, which includes SCAN, EKDIST, and HJCFIT (http:// www.ucl.ac.uk/Pharmacology/dcpr95.html); QuB (www.qub.buValo.edu), Pulse/ Patchmaster (HEKA Elecktronik), Tac (Bruxton Inc.), and the Strathclyde Electrophysiology Software (http://spider.science.strath.ac.uk/sipbs/software_ses.htm). We use ClampFit and QuB for analyzing records (Dellis et al., 2006; Rahman et al., 2009).
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Drifting baseline in current traces is first checked and corrected manually using ClampFit. Current-amplitude histograms of recordings with no obvious sub-conductance states are measured with a half-amplitude threshold-crossing criterion using ClampFit (Sachs et al., 1982; Colquhoun, 1994). Only events lasting longer than twice the filter rise time (tr ¼ 0.3321/fc, where fc is the cutoV frequency of the filter) can reach their full amplitude and so be reliably measured in a thresholdcrossing-based idealization procedure (Colquhoun, 1994) (Fig. 4A and B). In recordings with 1-kHz filtering, the predicted filter rise time (assuming the filter behaves as a Gaussian filter) is 332 ms. Events lasting < 1 ms are, therefore, omitted from the amplitude histograms. The peaks of the binned current amplitude histograms are fitted in ClampFit by sums of the appropriate number of Gaussian
10 pA
A
C 100 ms
Number of events
O
C O
B
1000
500
0
C
0
D Count/total
Count/total
0.06
to = 10 ms
0.06
0.03
0 0 1 2 Log (duration, ms)
tc1 = 1.14 ms (88%)
0.04 tc2 = 92 ms (12%)
0.02 0
1
5 10 Amplitude (pA)
−1
0 1 2 Log (duration, ms)
Fig. 4 Analysis of the behavior of single IP3R from nuclear patch-clamping recording. (A) Fragment of a raw record from an excised lumen-out nuclear patch with a single functional IP3R3. The recording conditions were identical to those shown in Fig. 3B. (B) The idealized record of the same trace produced as described in the text (Section IV.E). This idealized record is used for all subsequent analyses. (C) Allpoints current amplitude histogram, showing two peaks, one at 0 pA (the closed state) and a second at 10 pA (the single open state of one IP3R). (D) Distribution of the open (top) and closed (bottom) lifetimes shown as Sigworth–Sine plots (Sigworth and Sine, 1987). The plots suggest a single open state with to of 10 ms, and two closed states with tc of 1 and 92 ms.
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probability density functions (pdfs) (Fig. 4C). Mean current amplitudes are plotted against the corresponding applied potentials to create I–V curves (see Fig. 5). The unitary conductance (g) and reversal potentials (Erev) are derived by linear least-square regression analysis using statistical package such as Prism 5 (GraphPad Software, Inc.) or Origin Pro 7.5 (OriginLab Corporation). Because g is a fundamental property of any ion channel, reflecting interactions between permeating ions and the residues that form the pore and lead to it, I–V relationships are often used as ‘‘fingerprints’’ to help identify channels. With Kþ as charge carrier, the I–V relationships of mammalian IP3R in excised nuclear patches from DT40-KO cells are linear across a range of applied potentials ( 60 to þ 60 mV) (Fig. 5). From the slopes of these I–V plots, we have consistently observed two populations of IP3R with unitary Kþ conductances (gK) of either 120 or 200 pS (Dellis et al., 2006; Rahman and Taylor, 2009; Rahman et al., 2009) (Fig. 5B). Neither current was detected in the nuclear envelope of DT40-KO cells or from DT40-KO cells stably expressing IP3R in the absence of IP3, or with IP3 in the presence of a competitive antagonist. These values of gK are lower than reported ( 320–360 pS) for IP3R in the nuclear envelope of mammalian cells (Foskett et al., 2007), but there is wide variation in published values (from 9 to 480 pS) (Cheung et al., 2010; Rahman and Taylor, 2009). The reason for these disparities is unresolved, but it may reflect variable amounts of free Mg2þ in PS causing a reduction in gK (Mak and Foskett, 1998; Rahman and Taylor, 2009). It is, however, clear from analyses of I–V relationships that all IP3R have a large g for monovalent cations and lesser g for bivalent cations (Dellis et al., 2006; Foskett et al., 2007) (Fig. 5B). A
B
20 pA
15
500 ms +60 mV
I (pA)
10
C
5 +40 mV
C
−80
−40
−5
0 mV
C
−40 mV
−10
C
−15
−60 mV
C
40
80
V (mV)
Fig. 5 Current–voltage relationship for nuclear IP3R. (A) Currents were recorded from lumen-out
patches excised from the nucleus of DT40-KO cells stably expressing IP3R3. PS included IP3 (10 mM), ATP (5 mM), and a free [Ca2þ] of 200 nM. Kþ was the charge-carrier and the holding potential was varied between þ60 and 60 mV as shown. C denotes the closed state. (B) From the slope of the current–voltage (I–V) relationship, g was 208 pS. Results are means SEM, n ¼ 4.
8. Patch-Clamp Recording of IP3 Receptors
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Mutations within the putative pore region of IP3R that change g provide direct evidence that the residues within the P-loop linking the last pair of transmembrane domains are likely to contribute to the ion-permeation pathway (Boehning et al., 2001b; Schug et al., 2008). Future work along similar lines is likely to define more precisely the structural determinants of ion permeation. In addition, our demonstration that similar point mutations aVected g of the IP3-activated currents detected in the plasma membrane of DT40 cells expressing mutant IP3R provided definitive evidence that the currents were carried directly by IP3R, rather than by another plasma membrane channel with which IP3R in the ER might have associated (Dellis et al., 2006). Resolving the unitary current events associated with opening of individual IP3R also allows functional IP3R to be counted. The number of active IP3R in a patch can be estimated from the maximal number of simultaneous openings to the unitary current level (Horn, 1991) (Figs. 1C and 4C). The likelihood of several channels opening simultaneously depends upon their Po and the number of channels (N). We would, for example, need to wait much longer, on average, for six IP3R with low Po to open simultaneously than for the simultaneous opening of two IP3R with high Po. We can be confident (p < 0.01) that we have detected the entire complement of active IP3R within a patch, when the recording period is longer than 5(sNþ 1) (Ionescu et al., 2006), where " # to NtD ð1Þ sN ¼ exp to N ðPo ÞN and sN is the mean interval between successive simultaneous openings of all N IP3R; tD the minimum duration of an open event detectable after filtering (200 ms in our experiments); and to is the mean channel open time. Confidently, estimating the number of active IP3R within a patch is important, not the least because there has been a suggestion that increasing concentrations of IP3 cause increases in both Po (making it easier to detect simultaneous openings) and the number of active IP3R (Ionescu et al., 2006). This interesting and unprecedented behavior, which we fail to see (Rahman et al., 2009), has been invoked to explain the unusual pattern of quantal Ca2þ release observed for IP3R (Taylor, 1992). By varying the concentrations of cations on either side of the membrane (Section IV.D and Fig. 6A), the relative permeability (PBa/PK) can be calculated using a modified version of the Goldman–Hodgkin–Katz (GHK) equation (Bezprozvanny and Ehrlich, 1994; Fatt and Ginsborg, 1958): 2þ RT 4PBa Ba o ln ð2Þ Erev ¼ 2F PK ½Kþ i where PBa/PK is the relative permeability to Ba2þ and Kþ, [Kþ]i the [Kþ] in PS, [Ba2þ]o the [Ba2þ] in BS, Erev the reversal potential (corrected for the LJP, see Section IV.D), R the universal gas constant, F the Faraday constant, and T is the
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A
B
5 pA
+40 mV 500 ms 5
C
I (pA)
+60 mV −80
−40
40
C
80
V (mV)
−60 mV C −5
Fig. 6 Determining the cation-selectivity of IP3R from nuclear patch-clamp recording. (A) Currents were recorded in the same way as described in Fig. 5A, but with the usual PS changed to include Ba2þ (50 mM) rather than Kþ. The currents recorded at diVerent holding potentials are shown. C denotes the closed state. (B) I–V relationship showing a reversal potential (Erev) of 23.8 1.4 mV after correction for the liquid junction potential (Section IV.D). From the modified GHK equation, this suggests that the permeability to Ba2þ relative to Kþ (PBa/PK) is 4.7. The unitary conductance (g) from the slope of the plot is 45 4 pS.
absolute temperature (K). These analyses have established that IP3R (Dellis et al., 2006; Foskett et al., 2007), like ryanodine receptors (Williams, 2002), are far less selective (PBa/PK 7) than Ca2þ channels in the plasma membrane (Fig. 6). The distinction is important because channels within the plasma membrane must be able to discriminate between the many ions with an electrochemical gradient across the membrane, whereas Ca2þ is probably the only cation with an appreciable gradient across the ER membrane (Somlyo et al., 1977). In addition to revealing the properties of the open pore, single-channel analyses can also shed light on the steps that lead to its opening. The kinetic analyses of singlechannel records described here require that channel behavior has attained a steadystate. This is most easily assessed from a stability plot of single-channel open probability (Po) versus time (Colquhoun, 1994; Weiss and Magleby, 1989). Only records or parts thereof with an overall steady-state Po should be used for kinetic analysis. Files with stable baselines are exported as QuB-supported file formats (.ldt). In QuB, the files are further examined and sections of data with spurious noise are excluded using the preprocessing module (‘‘Pre’’). Current traces are then idealized into noise-free, open, and closed transitions using the segmental k-means (SKM) algorithm in the QuB suite. This uses a hidden Markov model (HMM) to decide whether each excursion in the record should be classified as an open or closed state based upon its amplitude (Qin, 2004) (Fig. 4B). The output at this stage is a categorization of every transition into a switch between current amplitudes: a single closed current amplitude (baseline noise) and one or several amplitudes of the open channel (s). Where several evenly spaced current amplitudes are detected, it can be diYcult to resolve whether they arise from openings of several channels or switches between
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equally spaced sub-conductance states of a single channel (Rahman and Taylor, 2009). For IP3R, sub-conductance states are rare (Rahman and Taylor, 2009), allowing the simplest possible scheme, a switch between a single closed (C) and open (O) state (C $ O) with arbitrarily chosen rate constants (e.g., 100 s 1), to be used for the initial idealization (Qin, 2004). Beginning with this simple scheme does not compromise later analyses that might reveal more complex relationships between several open and closed states. Direct comparison of raw traces with their idealized versions is essential at this stage to confirm the fidelity of the idealization procedure. Hitherto, the analysis, has considered only the amplitudes of the currents, the next step considers the durations of these events in records from single channels. This provides the opportunity to resolve diVerent open and closed states and possible relationships between them, leading to plausible gating schemes. The distribution of lifetimes of a single state of a channel is described by a single exponential (Colquhoun, 1994). The analysis attempts iteratively to establish, for each potential gating scheme (beginning with the simplest, C $ O), the number of exponential functions required to describe the closed and open lifetimes derived from the idealization procedure. A maximum interval likelihood method (MIL) is used to fit the lifetimes with pdfs (Qin et al., 1996, 1997, 2000). During this fitting process, a dead-time of 200 ms (twice the sampling interval) is retrospectively imposed for the correction of missed events (Sivilotti, 2010). Dwell-time histograms are generated and displayed with logarithmic abscissa and square root ordinate (Fig. 4D) (Sigworth and Sine, 1987) and fitted by a mixture of exponential pdfs, defined in the function f(t) as fðtÞ ¼
n X ai i¼1
ti
expðt=ti Þ
ð3Þ
where ai is the fractional area occupied by the ith component in the distribution, such that the areas corresponding to all components sum to unity, and ti is the time constant for the ith component. The mean life-time (t) is given by the following equation: t¼
n X
ðai ti Þ
ð4Þ
i¼1
The Sigworth–Sine transformation (Fig. 4D) allows a single plot clearly to display dwell-times spanning several orders of magnitude. Individual exponential components of the distribution can be directly identified from the peaks of the distribution. After iterative exploration of alternative gating schemes, the log likelihood ratio (Colquhoun, 1994) is used to identify the scheme that best fits the data. The chosen scheme is then used to reidealize the raw data to provide the final gating parameters (mean life-times and Po). Although these are the methods we have used to address the gating of IP3R (Rahman et al., 2009), more sophisticated approaches exploit the additional information that lurks in the correlations that exist between transitions (McManus et al., 1985).
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Analyses like these identify the numbers of stable open and closed states and plausible relationships between them. They lead thereby to models of the steps through which the IP3R passes between its inactive and open states. Such analyses have so far been rather limited for IP3R, but they clearly suggest the existence of a single open state and several closed states (Ionescu et al., 2007; Rahman et al., 2009) (Fig. 4D). Extending the analysis to patches, in which we detected several IP3R, allowed us to demonstrate that IP3 causes IP3R to form small clusters of 4–5 channels within which to is reduced from 10 to 5 ms (Rahman et al., 2009). These observations lead us to suggest that IP3 contribute to the evolution of elementary Ca2þ signals by both regulating IP3R activity and by assembling IP3R into clusters, within which regulation of IP3R by Ca2þ and IP3 is retuned (Rahman and Taylor, 2009; Rahman et al., 2009; Taylor et al., 2009a). For most channels, including IP3R, single-channel open probability (Po) (rather than g or the number of active channels) is the behavior that changes as the stimulus intensity varies. Increasing IP3 or Ca2þ increases Po of IP3R because both ligands shorten the duration of the closed times, without aVecting to; hence, the probability of finding the channel open (Po) is increased (Foskett et al., 2007; Rahman et al., 2009). Po is calculated from the fitted amplitude histograms of the current traces (typically lasting 1 min for IP3R) (Ding and Sachs, 1999): Po ¼
Ao Ao þ Ac
ð5Þ
where Ao and Ac are the areas under the curves corresponding to the open and closed states in the current amplitude histogram, respectively. When IP3R activity is low, it becomes very diYcult to know how many channels are contributing because it is unlikely that all will open simultaneously. Under these conditions, the overall activity is better expressed as NPo which is defined as (Ching et al., 1999; Rahman et al., 2009): N P ðntn Þ
NPo ¼
n¼1
T
ð6Þ
where tn is the total time for which n IP3R are simultaneously open and T is the duration of the recording.
V. Concluding Remarks Patch-clamp recording of IP3R expressed within the nuclear envelope allows single-channel analyses of these otherwise inaccessible intracellular Ca2þ channels (Figs. 1 and 2). DT40-KO cells provide a null background (Fig. 3) for expression of recombinant and mutant IP3R allowing functional analysis of IP3R with defined
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composition (Taylor et al., 2009b; Tovey et al., 2006). Nuclear patch-clamp recording of DT40 cells heterologously expressing mammalian IP3R, therefore, allows single-channel recording with its exquisite temporal resolution to be combined with opportunities to manipulate systematically the structure of the expressed IP3R. The stability of these patch-clamp recordings in a native membrane and the opportunity to apply them in various configurations (Fig. 2) aVord valuable opportunities to examine the behavior of small numbers of IP3R directly (Rahman et al., 2009) and as a means to address the mechanisms underlying IP3R activation (Rossi et al., 2009). In the short period during which nuclear patch-clamp analyses have been applied to IP3R, they have succeeded in confirming that IP3R are large conductance, relatively nonselective cation channels, and revealed the durations of the channel openings and closing (Dellis et al., 2006; Foskett et al., 2007; Rahman et al., 2009) (Figs. 4–6). Together, these insights allow estimates of the likely Ca2þ fluxes through individual IP3R for comparison with optical measurements of the elementary Ca2þ signals evoked by IP3 in situ (Shuai et al., 2007, 2008). Combining site-directed mutagenesis with nuclear patch-clamp recording has provided direct evidence that the pore of IP3R is formed by residues within the ‘‘P-loop’’ linking the final pair of transmembrane domains of each IP3R subunit (Boehning et al., 2001b; Dellis et al., 2006, 2008; Schug et al., 2008). The eVects of a novel family of synthetic partial agonists on normal and mutant IP3R analyzed by nuclear patchclamp recording have shed light on the first stages of IP3R activation by showing that the initial conformation changes evoked by IP3 binding to the IP3-binding core pass onward toward the pore entirely via the N-terminal suppressor domain (Rossi et al., 2009). Similar analyses have revealed the means, whereby ATP (Betzenhauser et al., 2008b, 2009b), cyclic AMP-dependent protein kinase (Betzenhauser et al., 2009a), cyclic AMP (Tovey et al., 2010), and various accessory proteins (Cheung et al., 2008, 2010; Li et al., 2007) modulate IP3R behavior. Future application of the nuclear patch-clamp technique to IP3R is certain to add further to our understanding of the stochastic behavior of single and clustered IP3R and to resolving the structural basis of IP3R activation. Acknowledgments This work was supported by grants from the Wellcome Trust, and the Biotechnology and Biological Sciences Research Council (UK). T. R. is a Drapers’ Company Research Fellow at Pembroke College, Cambridge.
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Taufiq Rahman and Colin W. Taylor Barry, P. H. (1994). JPCalc, a software package for calculating liquid junction potential corrections in patch-clamp, intracellular, epithelial and bilayer measurements and for correcting junction potential measurements. J. Neurosci. Methods 51, 107–116. Berridge, M. J., Lipp, P., and Bootman, M. D. (2000). The versatility and universality of calcium signalling. Nat. Rev. Mol. Cell Biol. 1, 11–21. Bers, D. M., Patton, C. W., and Nuccitelli, R. (1994). A practical guide to the preparation of Ca2þ buVers. In ‘‘Methods in Cell Biology,’’ (R. Nuccitelli, ed.), Vol. 40, pp. 4–29. Academic Press, San Diego, CA. Betzenhauser, M. J., Wagner, L. E., II, Iwai, M., Michikawa, T., Mikoshiba, K., and Yule, D. I. (2008). ATP modulation of Ca2þ release by type-2 and type-3 InsP3R: DiVering ATP sensitivities and molecular determinants of action. J. Biol. Chem. 283, 21579–21587. Betzenhauser, M. J., Wagner, L. E., II, Won, J. H., and Yule, D. I. (2008). Studying isoform-specific inositol 1, 4, 5-trisphosphate receptor function and regulation. Methods 46, 177–182. Betzenhauser, M. J., Fike, J. L., Wagner, L. E., II, and Yule, D. I. (2009). Protein kinase A increases type-2 inositol 1, 4, 5-trisphosphate receptor activity by phosphorylation of serine 937. J. Biol. Chem. 284, 25116–25125. Betzenhauser, M. J., Wagner, L. E., II, Park, H. S., and Yule, D. I. (2009). ATP regulation of type-1 inositol 1, 4, 5-trisphosphate receptor activity does not require walker A-type ATP-binding motifs. J. Biol. Chem. 284, 16156–16163. Bezprozvanny, I., and Ehrlich, B. E. (1994). Inositol (1, 4, 5)-trisphosphate (InsP3)-gated Ca channels from cerebellum: Conduction properties for divalent cations and regulation by intraluminal calcium. J. Gen. Physiol. 104, 821–856. Bezprozvanny, I., Watras, J., and Ehrlich, B. E. (1991). Bell-shaped calcium-response curves for Ins(1, 4, 5)P3- and calcium-gated channels from endoplasmic reticulum of cerebellum. Nature 351, 751–754. Boehning, D., and Joseph, S. K. (2000). Direct association of ligand-binding and pore domains in homo- and heterotetrameric inositol 1, 4, 5-trisphosphate receptors. EMBO J. 19, 5450–5459. Boehning, D., Joseph, S. K., Mak, D.-O. D., and Foskett, J. K. (2001). Single-channel recordings of recombinant inositol trisphosphate receptors in mammalian nuclear envelope. Biophys. J. 81, 117–124. Boehning, D., Mak, D.-O. D., Foskett, J. K., and Joseph, S. K. (2001). Molecular determinants of ion permeation and selectivity in inositol 1,4,5-trisphosphate receptor Ca2þ channels. J. Biol. Chem. 276, 13509–13512. Bootman, M. D., and Berridge, M. J. (1995). The elemental principles of calcium signaling. Cell 83, 675–678. Bootman, M. D., Berridge, M. J., and Lipp, P. (1997). Cooking with calcium: The recipes for composing global signals from elementary events. Cell 91, 367–373. Bosanac, I., Alattia, J.-R., Mal, T. K., Chan, J., Talarico, S., Tong, F. K., Tong, K. I., Yoshikawa, F., Furuichi, T., Iwai, M., Michikawa, T., Mikoshiba, K., et al. (2002). Structure of the inositol 1, 4, 5trisphosphate receptor binding core in complex with its ligand. Nature 420, 696–700. Bosanac, I., Yamazaki, H., Matsu-ura, T., Michikawa, M., Mikoshiba, K., and Ikura, M. (2005). Crystal structure of the ligand binding suppressor domain of type 1 inositol 1, 4, 5-trisphosphate receptor. Mol. Cell 17, 193–203. Buerstedde, J. M., and Takeda, S. (1991). Increased ratio of targeted to random integration after transfection of chicken B cell lines. Cell 67, 179–188. Bustamante, J. O. (1994). Nuclear electrophysiology. J. Membr. Biol. 138, 105–112. Bustamante, J. O., and Varanda, W. A. (1998). Patch-clamp detection of macromolecular translocation along nuclear pores. Braz. J. Med. Biol. Res. 31, 333–354. Cardy, T. J. A., Traynor, D., and Taylor, C. W. (1997). DiVerential regulation of types 1 and 3 inositol trisphosphate receptors by cytosolic Ca2þ. Biochem. J. 328, 785–793. Cheung, K. H., Shineman, D., Muller, M., Cardenas, C., Mei, L., Yang, J., Tomita, T., Iwatsubo, T., Lee, V. M., and Foskett, J. K. (2008). Mechanism of Ca2þ disruption in Alzheimer’s disease by presenilin regulation of InsP3 receptor channel gating. Neuron 58, 871–883.
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CHAPTER 9
Confocal and Multiphoton Imaging of Intracellular Ca2þ Godfrey Smith,* Martyn Reynolds,† Francis Burton,* and Ole Johan Kemi* *School of Life Sciences University of Glasgow United Kingdom †
Cairn Research Limited Faversham, Kent United Kingdom
I. II. III. IV. V. VI. VII. VIII. IX. X. XI. XII. XIII. XIV. XV. XVI. XVII. XVIII. XIX. XX.
Abstract Why Study Ca2þ Signaling with Confocal and Multiphoton Microscopy Confocal Microscopy Limitations in Speed of Confocal Imaging Laser Scanning Confocal Microscopy Total Internal Reflection Fluorescence Microscopy Fo¨rster Resonance Energy Transfer Microscopy Parallel Scanning Confocal Systems Spinning Disk Confocal Microscopy Programmable Matrix Microscopy Advantages and Disadvantages of Confocal Microscopy Multiphoton Excitation Laser Scanning Microscopy Ca2þ Indicators for Use in Confocal and Multiphoton Microscopy Use of Dyes for Single-Photon Confocal Microscopy Use of Dyes for 2P Excitation Microscopy Is It Worth Converting the Intracellular Fluorescence Signal to [Ca2þ]? Calibration of Single Wavelength Dyes Estimation of Fmax Values Estimation of Fmin or the Dynamic Range of the Dye Consequence of Errors in Estimation of Intrinsic and Dye Fluorescence Multimodal and Multiple Fluorophore Confocal and Multiphoton Microscopy References
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0091-679X/10 $35.00 DOI: 10.1016/S0091-679X(10)99009-2
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Abstract This chapter compares the imaging capabilities of a range of systems including multiphoton microscopy in regard to measurements of intracellular Ca2þ within living cells. In particular, the excitation spectra of popular fluorescent Ca2þ indicators are shown during 1P and 2P excitation. The strengths and limitations of the current indicators are discussed along with error analysis which highlights the value of matching the Ca2þ aYnity of the dye to a particular aspect of Ca2þ signaling. Finally, the combined emission spectra of Ca2þ and voltage sensitive dyes are compared to allow the choice of the optimum combination to allow simultaneous intracellular Ca2þ and membrane voltage measurement.
I. Why Study Ca2þ Signaling with Confocal and Multiphoton Microscopy Ca2þ is a ubiquitous intracellular messenger that controls a large number of cellular processes, such as gene transcription, excitation, contraction, apoptosis, cellular respiration, and the activity levels of many cell-signaling messenger cascades. Inside the cell, Ca2þ may, under various conditions, sequester into the sarco/ endoplasmic reticulum, mitochondria, and the nucleus, or exist in the cytosol either in its free form or as bound to buVers. Typically, a large Ca2þ concentration gradient is maintained across the plasma membrane of the cell. Because of diVerent Ca2þ channels, pumps, and exchangers on the membranes of the cell or organelles, Ca2þ fluxes may be created at multiple locations in the cell. Therefore, Ca2þ concentration and signal may be specific with respect to both location and time. Moreover, Ca2þ may concentrate in distinct cytoplasmic regions because of tight physical loci not enclosed by membranes, for example, the dyadic area between the transverse tubule and the sarco/endoplasmic reticulum of muscle cells. Given the large number of Ca2þ channels feeding it with Ca2þ from both the extracellular space (transverse tubule) and the sarco/endoplasmic reticulum, such that the dyad may transiently have very diVerent localized Ca2þ concentrations compared to the rest of the cytosol which may be only nanometers away. Thus, Ca2þ localizes in the cytosol as well as within organellar compartments, and these Ca2þ signals may last for very short timeframes (ns) or for substantially longer periods of time (min). Ca2þ signaling per se is not within the remit of this chapter and will not be covered in any detail, but the interested reader is referred to other sources, for example, Bootman et al. (2001). Nonetheless, for the purpose of this chapter, it is important to acknowledge that the average Ca2þ concentration in any given cell usually ranges 0.01–1 mM, but that the Ca2þ almost never exists uniformly across the cell, and that local Ca2þ events may occur with very fast time courses. This therefore requires Ca2þ imaging of live specimens with high spatial and temporal resolution. Thus, one would
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ideally want to distinguish Ca2þ in distinct areas that may be within a nanometer distance from each other, and to record localized Ca2þ events that may last only a millisecond. Although such requirements tax any given microscopy system, confocal and multiphoton microscopy systems oVer a range of imaging capabilities that fulfill these criteria.
II. Confocal Microscopy Optical sectioning by confocal microscopy adds several benefits to Ca2þ imaging. Since its early development, confocal microscopy has fundamentally transformed optical imaging to now provide a valuable addition that allows unprecedented imaging of minute optical sections within live specimens in close to real-time speeds. In terms of Ca2þ imaging, this has opened up new fields of study, given that Ca2þ signaling in many, if not all, biological systems is compartmentalized within small sections of the cell and occurs often at very high velocities. Confocal microscopy allows the study of these events within discrete depths of cell or tissue by blocking light originating outside the plane of focus. This is achieved by the addition of confocal apertures in front of the illumination source and in the image plane directly in front of the signal detection system (see Fig. 1); usually, a photomultiplier tube (PMT) that rejects out-of-focus light originating from fluorescence outwith the area of interest (the focal plane), and only allowing in-focus light through to the PMT (Webb, 1999) (Fig. 1). This is in contrast to regular epifluorescence microscopy, in which the majority of the fluorescence is out-of-focus light that generally reduces the contrast of the in-focus light, and also dramatically compromises in-focus detail. This occurs since the emitted fluorescence cannot be discriminated along the Z-axis (top to bottom), and also less along the X- and Y-axes (although this has also to do with excitation light sources; see later) in conventional epifluorescence microscopy (Lichtman and Conchello, 2005) (See also later). Thus, although confocal microscopy also excites the specimen along the entire Z-axis in line with conventional epifluorescence microscopy, only in-focus light is allowed to pass the pinhole of the confocal aperture to enter the signal detector. Importantly, confocal imaging may be performed on live specimens residing under physiologic conditions and that are electrically, chemically, mechanically, and otherwise active and healthy. Specimens may also be electrically and mechanically stimulated and superfused by any given solutions that would not interfere with the confocal imaging. The diVerence between regular epifluorescence and confocal microscopy light capture abilities can be illustrated by the following examples. Considering that the depth of focus of a high numerical aperture (NA > 1.3) objective is restricted to 0.3 mm, whereas the depth of a fluorescent cell may be 5–25 mm, it becomes clear that the depth of focus will only constitute 1–5% of the full depth of the cell. Since epifluorescence microscopy captures light along the entire Z-axis, 95–99% of the
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Light detector Confocal aperture with pinhole
Laser Dichroic beamsplitter
Objective
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Fig. 1 Overview of the optical pathway of a confocal microscope. The blue path illustrates the excitation light, whereas the green path illustrates the emitted fluorescence light. Note that the confocal aperture with the pinhole in front of the light detector (usually, a photomultiplier tube (PMT)) blocks out-of-focus light.
cell volume will contribute to unwanted out-of-focus background signal, or noise (Lichtman and Conchello, 2005), and this cannot be distinguished from in-focus fluorescence. Thus, most of the cell will be out of focus, and with it, the vast majority of the signal will come from out-of-focus areas. In contrast, setting the pinhole of the confocal aperture to 1 airy unit to achieve true confocality will provide an optical section or Z-resolution of 0.5–1 mm with the same high NA objective as described above. This will only allow a minimum of out-of-focus light to reach the signal detector, without any other interference to the optical pathway or any secondary digital processing of the signal at the time of recording apart from the scanning and building of the image itself, which otherwise would have further compromised the scanning speed. The full 3D XYZ-resolution, or the ability to discern two points from each other, will, however, be diVraction-limited as determined by the point spread function (PSF) set by the optical performance of the microscope. With high NA objectives (> 1.2), this is typically 0.3 0.3 0.6 mm (Cox and Sheppard, 2004). Although Z-resolution is dramatically diVerent between conventional epifluorescence and confocal microscopes, the 2D spatial resolution in the XY-field is not, though factors, such as excitation wavelength, objectives and the optical
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pathway, and diVerent media and surfaces, will also aVect this. However, several factors associated with the confocal principle allow for improving also XYresolution, as compared to widefield epifluorescence microscopy. First, spatial resolution may be improved by further reducing the pinhole diameter in the confocal aperture to a size smaller than the width of the central disk of the airy unit pattern, though this also dramatically reduces light transmission. This principle works because the pinhole is aligned with the center of the airy unit pattern of the illuminating beam, which means that any emission originating from any fluorescent molecules excited by the outer airy rings of the illuminating beam will be blocked by the confocal aperture; like all light, the illumination beam also presents with a airy wave pattern consisting of a central bright spot and outer ring waves that in comparison are more faint. In other words, the resultant fluorescence emission may be experimentally manipulated to originate from an area smaller than the airy unit, which cannot be achieved by conventional widefield epifluorescence microscopy. Moreover, because the PSF of the confocal microscope is narrower at normalized light intensities relative to that of the conventional widefield microscope, it means the XY spatial resolution will be 1.4 greater with a confocal microscope than a widefield microscope (Conchello and Lichtman, 2005). Finally, some of the in-focus light will scatter on its way through the specimen, due to diVraction, reflection, and refraction as cell structures interfere with the light path. This also compromises fluorescence, but not confocal microscopy, as the confocal aperture also blocks scattered light from reaching the signal detector. In addition, combining confocal microscopy with total internal reflection fluorescence (TIRF) or Fo¨rster resonance energy transfer (FRET) microscopy techniques has the capacity to increase resolution to only a few tens of nanometers (see below for more detailed information). Other techniques such as narrowing the boundaries of the PSF by suppressing (de-exciting) the fluorescence from the edge of the center spot of the airy pattern by stimulated emission depletion (STED), and other nonlinear optical masking techniques, have further enhanced optical resolution of confocal microscopes (Bullen, 2008; Willig et al., 2006), though these techniques are not yet compatible with fast scanning of Ca2þ events that take place over fast timescales, and will therefore not be discussed here. Finally, secondary signal processing or deconvolution (computationally reverse optical distortion to enhance resolution) of the recorded images also serve to enhance spatial resolution of both confocal and epifluorescence microscopy by a factor of 2–3. Because light scattering increases proportionally to increasing thickness of the specimen, this becomes more of an issue with deeper imaging of thicker specimens. Therefore, appropriately setting the pinhole not only allows for imaging of thin optical sections, but also aVects the signal-to-noise (SNR) ratio. Whereas a case made be made that reducing the pinhole diameter increases XYZ-resolution (especially Z-, but also XY-resolution; see above), opening the pinhole to approximately match the projected image of the diVraction-limited spot (1.22l/NA,
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where l is the illumination wavelength) will substantially increase the SNR with only minimal reduction in the Z-resolution (Conchello et al., 1994). Thus, this would increase the quality of the signal with little degradation of the depth discrimination.
III. Limitations in Speed of Confocal Imaging The removal of out-of-focus light allows for relatively fast imaging of 0.6 mm thin sections either in spot, line, or frame modes that either may be repeated sequentially, or combined with stepwise up- or down-focusing through the specimen in order to generate 3D reconstructions. 3D sectioning does not allow for recording of cellular events in real time, but repeated 1D line imaging or even repeated 2D imaging with reduced frame sizes or restricted pixel numbers in contrast do allow for relatively fast recording with a temporal resolution of approaching a microsecond scale. Although not as fast as regular widefield imaging, even 2D frame imaging may still be acquired on fast time scales, usually within hundreds of millisecond, though there will be a trade-oV between temporal and spatial resolution. The reason for the lower temporal resolution compared to widefield imaging is that conventional confocal imaging requires some form of scanning, that is, sequential pixel sampling, in order to ‘‘build’’ an image, which thus happens pixel-by-pixel. In contrast, the whole field during widefield imaging is captured simultaneously either by PMTs or charge-coupled device (CCD) cameras (Ogden, 1994). For some Ca2þ events, imaging with a temporal resolution in the order of milliseconds may be satisfactory, but other events may occur considerably faster than this. Likewise, some events allow the microscopist to sample images with a low spatial resolution, whereas others require the opposite. Thus, the speed of confocal image acquisition depends on the mode and the settings of the scanning and how many pixels are scanned before returning to the same pixel again. This will be discussed later.
IV. Laser Scanning Confocal Microscopy Out-of-focus light rejection and image acquisition through a confocal aperture with a pinhole is the common principle that constitutes confocal microscopes, but the illumination and excitation principles may diVer between various systems. First, confocal microscopy by laser scanning the specimen (laser scanning confocal microscopy, often abbreviated to LSM or LSCM) is the most widely used illumination and excitation method today. During LSCM, a laser beam is directed on to the specimen, whereupon it scans the designated field, which may be a single spot (in reality rarely used for biological imaging apart from fluorescence recovery after photobleaching (FRAP) applications), a 1D line, or a 2D frame. The laser is controlled by the use of two oscillating
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mirrors in the scanhead that deflect the beam along a fast and slow axes perpendicular to one another. Thus, during a 2D frame scan, the beam is first directed along the horizontal axis, after which it ‘‘jumps’’ down one pixel and scans the next line, and this process continues until a full frame has been scanned. This may then be repeated for serial frame scanning, or the plane of focus may be moved along the Z-axis for 3D imaging. The fluorescence emission returns along the same light path (descanning), but is deflected by a dichroic mirror splitting the excitation and emission lights, such that the emission light only is directed onto the confocal aperture, where the in-focus light penetrates though the pinhole to reach the signal detector. Unfortunately, scanning is a rate-limiting step for gaining fast images, especially in a 2D frame scan mode, because of the mechanical characteristics of the mirrors. A typical 512 512 pixel 2D frame may be scanned in 1 s, depending on the settings under which the scan is performed. When scanspeed is of concern, several approaches may be taken to increase this, for example, by linescanning instead of framescanning. In this configuration, the same line is scanned sequentially for a given period of time to allow detection of one or more events occurring within the time frame of the scan. Although this provides a high temporal resolution, the acquired information is limited to a one-pixel wide area of the cell, such that information about events occurring elsewhere in the cell is missed. Furthermore, reducing the pixel dwell time (the period over which each pixel is scanned) also increases scanspeed, but this also reduces the SNR. Reduced SNR may partially be compensated for by increasing the laser power, but this may present other problems such as photodamage to the specimen; especially in live cells, and photobleaching. Finally, the length of the line or the size of the frame to be scanned may also be reduced, or fewer pixels may be scanned during 2D frame scanning to yield the same eVect, and the scan may also be run in a bidirectional mode instead of unidirectional, though using two lines running in opposite directions may cause a slight oVset between them, which tends to blur the signal. More recently, several approaches have been taken to increase scanspeeds, in particular for 2D frame imaging, such as utilizing resonant oscillating mirrors in the scanhead instead of the more conventional galvanometer-driven mechanical mirrors. Other options include arranging prisms and acousto-optical deflectors into the excitation light path to illuminate the entire line simultaneously, instead of a pixel-by-pixel illumination applied by the conventional laser scanning microscopes described above. This means that the scanning in a 2D frame mode would only involve movement along one dimension (X), since the other dimension (Y) would all be scanned at once (simultaneously), and therefore, 2D frame scanning may be performed at linescan speeds or at speeds approaching video rates, if the emitted fluorescence is deflected onto a linear CCD camera, though PMT arrays may also be used. Single PMTs would, however, not be able to construct the images if lines instead of single pixels are scanned. The caveat with these approaches is that true confocality will be lost because the pinhole of the confocal aperture must be replaced by one or more longitudinal slit openings to accommodate the simultaneous scanning of lines (hence, this is also called slit
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scanning). Also, series of holographic or curved mirrors in the scanhead have also been utilized to scan more than one pixel at a time, but this has remained more of a rarity compared to the abovementioned microscopy modifications (Callamaras and Parker, 1999; Tsien and Bacskai, 1995). The aforementioned confocal scanning approaches depend on the use of singlephoton lasers as the source of illumination and excitation. These are based on the principle that a single photon provides enough energy to excite a single fluorescent molecule, that is, to ‘‘lift’’ it from a ground state to the ‘‘excited’’ state. The phase where the fluorophore is lifted to the excited state lasts for femtoseconds (10 15 s), whereas the fluorophore remains in the higher-energy excited state for picoseconds (10 12 s) where it undergoes internal conversion and starts to vibrate, which eVectively leads to dissipation of energy, such that it drops back to the ground state; measurable to a time scale of nanoseconds (10 9 s). When this happens, the fluorophore releases a photon that due of the loss of energy has a longer wavelength (less energy), and this is what creates the fluorescence emission that may be measured by signal detectors such as PMTs or CCD cameras. The diVerence between the excitation and emission spectra (emission wavelengths being longer than excitation wavelengths) is called the Stokes shift. The process of excitation and subsequent relaxation with photon release and fluorescence emission can be illustrated by a Jablonski diagram (Fig. 2), and is not restricted to confocal microscopy, but is in fact the basis for all fluorescence techniques including epifluorescence microscopy and spectroscopy. As detailed above, it is the volume of the recorded fluorescent emission that diVers between confocal and epifluorescence microscopy modalities, though the volume of excitation may also diVer, but this has to do with how much of the specimen is subjected to the illumination light. However, although the laser excites fluorophores along the entire Z-axis of the specimen (see also above and Fig. 3), peak excitation and as such peak brightness occurs at the focal plane, whereas out-of-focus excitation decreases with the square of the distance from the focal plane. This is because the laser excitation beam presents with an hourglass shape, with the ‘‘waist’’ of the hourglass coinciding exactly with the focal plane. Several laser lines have been developed that allow single-photon excitation of fluorescent Ca2þ indicators (fluorophores), in particular, the multiline argon ion (Ar-ion) laser that provides high-intensity light from the ultraviolet (UV) to the green spectrum ( 250–514 nm wavelengths), the single-line helium– neon (He–Ne) lasers that extend the covered spectrum to 633 nm, and argon– krypton (Ar–Kr) lasers that provide high-intensity light from blue to red wavelengths. Thus, these lasers are well suited for exciting the common Ca2þ indicators dyes and are also as such much used in Ca2þ signaling research. Recent developments in solid state and diode lasers have also added more choices for the microscopist. However, lasers will not be covered in detail here, but the interested reader will find a wealth of literature on this topic by searching the appropriate literature databases or microscopy textbooks, literature that covers the topic from both physics and biology perspectives.
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V. Total Internal Reflection Fluorescence Microscopy Confocal microscopy coupled to TIRF provides a very thin optical section of fluorescence excitation that allows imaging with low background noise and minimal out-of-focus fluorescence. This is because total internal reflection can only occur when the excitation light beam in a medium of high refractive index reaches an interface of a medium with a lower refractive index at an angle of incidence that is greater than the specific critical angle y. When the light is totally internally reflected, none of it penetrates the medium with the lower refractive index, and ideally, there is no net energy flux escaping the glass.
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Fig. 3 Total internal reflection fluorescence (TIRF) microscopy. (A) Overview including the incident and reflected laser light paths within the objective. (B) Once the incident light reaches a medium with a lower refractive index at an angle greater than the critical angle y, the incident light does not penetrate the specimen, but an electromagnetic field is created that penetrates up to ~100 nm above the surface, called an evanescent wave. This may excite fluorophores within the range of the evanescent wave.
However, the reflected light generates an electromagnetic field that penetrates beyond the interface and into the lower refractive index medium as an evanescent wave. This wave, with a wavelength similar to the excitation light beam, decreases exponentially with the distance into the medium. The penetration depth of the evanescent wave may be manipulated by changing the angle of incidence beyond the critical angle, which may be calculated accurately by knowing the angle of incidence, but will typically be limited to 100 nm or less (Cleemann et al., 1997; Mashanov et al., 2003). This allows for imaging of Ca2þ events occurring in close proximity to the plasma membrane of live cells. However, this does require that the cell is positioned within the evanescent wave and not above it on the glass surface, which may well be the case for the majority of the cell if the cell is of a certain size. In our experience, it requires an experimental eVort to physically position large cells within the evanescent wave and yet still maintain physiological conditions for the cell. This is required because the evanescent wave is generated from the glass surface and not from the boundary of the cell.
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VI. Fo¨rster Resonance Energy Transfer Microscopy Confocal microscopy coupled with FRET imaging has experienced something of a surge of interest recently from Ca2þ researchers, because of the development of Ca2þ-sensitive cameleons. The FRET process is based upon nonradiative energy transfer from a fluorophore in an excited state (‘‘donor’’) to another chromophore (‘‘acceptor’’) that usually is, but does not have to be, a diVerent fluorescent molecule within a range of 10–100 Angstroms (1–10 nm) (fluorophore) (JaresErijman and Jovin, 2003). Thus, when FRET occurs, it is not only the acceptor emission that will be recordable, but the measurable emission from the donor will also be greatly reduced because of the energy transfer. Also, the intensity of the two emission bands will depend on the distance between the two donor and acceptor fluorescent proteins or molecules even within the FRET distance. The closer the distance between the donor and acceptor, the greater the longer-wavelength emission (from the acceptor) and the less the shorter-wavelength emission (from the donor). The fluorescence emission from the acceptor is discernible from the donor emission due to the spectral redshift, such that the instrumental requirement is that the two emission bands must be separately recordable. The presence of the longerwavelength acceptor emission will confirm that the acceptor molecule is within the FRET distance of the donor, that is, within a distance of 10 nm. Likewise, the absence of it suggests that the physical distance between the donor and acceptor is larger than that. Traditionally, FRET imaging has been used to study protein–protein interactions by tagging diVerent proteins with fluorescent probes (e.g., yellow or green fluorescent proteins (YFP or GFP)) and thereby study whether they exist within or outwith the FRET distance. However, it is the recent development of cameleons that has moved FRET imaging to also become a sought-after technique with respect to studies of Ca2þ. Cameleons are genetically encoded fluorescent proteins that are sensitive to Ca2þ, in which a blue- or cyan-mutant of GFP (serving as the donor), calmodulin that can bind to Ca2þ, a calmodulin-binding peptide such as the calmodulin-binding domain of skeletal muscle myosin light chain kinase (the M13 peptide), and a long-wavelength yellow mutant or normal GFP (serving as the acceptor) are tandemly fused (Miyawaki et al., 1997). This configuration forms a stable and compact complex that remains intact once transfected into the target cell, and subsequent development has also improved the spectral properties and rendered the cameleons less susceptible to changes in pH (enhanced GFPs). In the absence of Ca2þ, the cameleon remains in its linear tandem configuration, whereby the two GFP mutants (donor and acceptor) at the two flanks of the tandem are too far apart to create FRET. However, when the concentration of free Ca2þ increases, calmodulin binds to Ca2þ and undergoes a conformational change that also leads it to bind and wrap around the M13 peptide, which creates a compact configuration of the cameleon and therefore brings the donor and acceptor GFP mutants to within a distance where energy transfer may occur
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with much greater eYciency. Thus, the presence of Ca2þ creates FRET by the cameleon, which may be readily detected. Moreover, because Ca2þ aYnity can be tuned by incorporating mutations into the calmodulin protein, cameleons may detect free Ca2þ concentrations in the range 10 nM–10 mM, and this has been done to visualize local Ca2þ signals in the nucleus, sarco/endoplasmic reticulum, and the cytosol, by transfecting chimeras of the cameleon that also have the appropriate localization signals encoded in the complementary DNA (Miyawaki et al., 1997). A typical example of a cameleon used for FRET imaging of Ca2þ has an excitation spectrum peak at 442 nm for the donor (blue mutant of GFP), with the associated emission peaking at 486 nm. This wavelength FRET-excites the GFP on the acceptor side, which then emits longer wavelength fluorescence with a peak at 530 nm (Fig. 4). Several protocols for detecting and measuring FRET eYciency have been developed, of which some are more applicable to imaging of Ca2þ signals than others. These include measuring donor quenching, that is, measuring the decrease in the emission from the donor fluorophore, which appears because some of the energy emitted by the donor fluorophore is used to excite the acceptor chromophore/fluorophore. This is done by taking the ratio between donor and acceptor fluorescence during FRET as the numerator, and the same ratio in the absence of FRET (i.e., by removing either of the donor or acceptor fluorophores) as the denominator. Like all ratiometric quantifications, this has the advantage that the measure becomes independent of local variations in fluorescence. The disadvantage is that both the donor and acceptor fluorophores may be quenched by other factors that would misrepresent the results. Another method for measuring FRET eYciency is by measuring donor quenching and acceptor photobleaching, that is, the intensity of the donor emission in the presence of an acceptor relative to the intensity of the donor emission in the absence of an acceptor. In practice, the latter is done by first photobleaching the acceptor by illuminating it with light at the peak excitation spectrum of the acceptor fluorophore before measuring the donor emission. This protocol relies on the fact that fluorescing itself causes the fluorophores to lose their ability to fluoresce, a process called photobleaching. However, because photobleaching may take many minutes to achieve (up to 20 min), this may become less available in live specimens. Finally, FRET combined with fluorescence lifetime imaging (FLIM) has introduced a robust option for Ca2þ measurements, because it largely is unaVected by experimental conditions such as fluorophore concentrations and excitation intensities. In this combined FRET–FLIM approach, the change in donor lifetime is measured in the presence and absence of an acceptor. The principle behind this is that the period of time the donor will fluoresce (i.e., the lifetime of the donor) depends on the presence or absence of an acceptor (Levitt et al., 2009). As described above, the measurement of donor emission in the absence of an acceptor may be done by first photobleaching the acceptor. However, once the photobleached control images have been captured, this protocol allows for detailed imaging of Ca2þ signals over a short time period.
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Fig. 4 Cameleon-based Fo¨rster resonance energy transfer (FRET). (A) Schematic of the cameleon in the absence and presence of Ca2þ; note the conformational change in the calmodulin (Cam) and the Cam-binding domain of myosin light chain kinase (M13) upon binding to Ca2þ that allows for FRET between the donor and acceptor green fluorescent protein (FP) mutants. (B) The relative emission intensities at different wavelengths indicate whether or not Ca2þ is present, and hence, FRET occurs.
Since the introduction of cameleons, derivatives have been developed that fuse Cam with the Cam-binding peptide M13 in a similar fashion as cameleons, but are based on single GFPs instead of two GFP mutants with diVerent spectral properties that necessitate Ca2þ imaging by FRET. These derivatives, called pericams, can therefore be imaged by standard epifluorescence or confocal microscopes, and present with broad Ca2þ sensitivities and diVerent localization sequences that allow for measurements of a range of Ca2þ concentrations within specific organelles and intracellular targets (Kettlewell et al., 2009; Nagai et al., 2001).
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VII. Parallel Scanning Confocal Systems The main limitation of the traditional laser scanning confocal microscope systems discussed so far is their low full frame temporal resolution. At typical rates of just a few Hertz, an increase of 1–2 orders of magnitude in the speed of image acquisition is necessary to decipher the fast Ca2þ dynamics of systems such as neuronal networks or cardiac and muscle tissues. Recent developments in parallel multispot confocal illumination devices have gone some way to addressing these concerns, providing high contrast optical sectioning with typical rates in the 10–100 Hz domain. The multispot system uses an aperture mask at an illumination plane conjugate with the sample, where multiple illumination points with nonoverlapping airy disk profiles are projected to the sample simultaneously. This illumination pattern is then changed in a sequential fashion such that every image point is uniformly illuminated in a given time interval. Two approaches have been used in recent years to successfully achieve this fast pattern change: the Nipkow spinning disk where a patterned disk is spun at high speeds, and programmable matrix systems (digital mirror and liquid crystal arrays) where individual pixels can be switched ‘‘on’’ and ‘‘oV’’ at very high speeds. The parallel illumination approach has the obvious advantage that all image points can be illuminated much faster than in a conventional single scan system, but the drawback is that a 2D imaging detector is required to record the image, which then becomes the limiting factor for capturing image frames. Limitations of the detectors (typically CCD cameras) are further exacerbated by the requirement for fast detection, as these devices have a noise floor below which the fluorescence signal cannot be resolved. The fundamental problem is that in order to increase frame rates, the exposure time of a frame has to be reduced, thus limiting the number of detectable photons in an integration interval. Simultaneously, the transfer rate of data from the camera has to be increased, a process by which the read noise of CCD cameras increases. Many of these concerns are resolved by modern high-end electron multiplication (EM) CCD cameras such as the iXon 897 (Andor), Evolve (Photometrics), or ImagEM (Hamamatsu). These EM-CCD cameras can amplify small photoelectron signals above the read noise of the camera; providing the sensitivity and speed required making these theoretical parallel approaches a practical reality.
VIII. Spinning Disk Confocal Microscopy These systems illuminate the specimen simultaneously with a large number of non-overlapping points of light by using multiple (hundreds to thousands) pinholes arranged in a geometrically precise spiral pattern on a spinning disk (Nipkow disk). The disk is placed at an image plane conjugate with the sample, and the illumination is filtered by this disk. Thus, the specimen is raster scanned rather than single-spot
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scanned, such that the entire 2D frame may be illuminated semi-simultaneously. Fluorescence emitted from each illumination point then returns through the same aperture of the mask to give a set of confocal points in the image. When the disk is spun at high speed (typically several thousand revolutions per minute), an apparently continuous image is obtained when viewed through an eyepiece. The resultant image is traversed with scan lines, but precise synchronization of the illumination and detection systems virtually eliminates this artifact in well configured systems (Wilson et al., 1996). Thus, this approach allows for very fast scan rates without significantly compromising the SNR (Wang et al., 2005). As the input illumination power is spread over a much larger area than with conventional scanning confocal systems, light throughput can be a serious impediment to successful implementation of spinning disk systems. This was partially resolved by enhancing the original design by means of a second disk spinning in sequence with the Nipkow pinhole array disk. This second disk is sited between the dichroic and the light source and contains a microlens array that maps a miniature lens to each pinhole (Tanaami et al., 2002), thus improving the illumination eYciency by focusing the lightbeam onto the pinhole (Fig. 5A). This also reduces the backscattering of light at the surface of the Nipkow disk, which substantially increases SNR. The emission detection pathway is not aVected by this modification. Use of specialist cameras and fast versions of the spinning disk head can now enable imaging rates of up to 2 kHz. However, the drawbacks are that the pinholes on the spinning disk are inflexible, and the dwell time per pixel is usually very short ( 100 ns), which may severely reduce the SNR, although the frequent illumination of the same pixel as the disk rotates may compensate for this eVect.
IX. Programmable Matrix Microscopy These instruments are based on the principle of spatially filtering full field illumination in a defined pattern at high speed, so as to give it a prescribed, dynamic structure. By using appropriate filtering patterns, the device can simulate the optical behavior of confocal scanning microscopes. These systems are directly comparable to the spinning disk approach in that the illumination device consists of an array of small apertures that act as both the illumination and detection pinholes. The principal diVerence, and advantage, is that the elements of the array are individually addressable, allowing far greater flexibility in experimental design compared to the spinning disk (Hanley et al., 1999). As a practical example, selectively and sequentially illuminating individual cells within the field is possible with the array system, whereas the Nipkow disk can only operate at full frame. Two technologies have been used in implementing practical programmable matrix systems. The first makes use of a digital micromirror device (DMD), an array of micrometer-sized mirrors whose angle can be independently controlled to direct illumination to an ‘‘on’’ (confocal) or ‘‘oV’’ (non-confocal) pathway.
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The alternative uses a reflective liquid crystal display (LCD), an array of micrometer-sized liquid crystal ‘‘pixels,’’ the base of which is coated with a reflective layer (Fig. 5B). Each element is individually addressable, creating a controlled polarization pattern that is optically transformed into a confocal intensity pattern. So far, the liquid crystal approach forms the basis for the commercial utilization of this technique. This allows for using both the confocal image (conjugate with the array
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mask) and nonconjugate image to provide further potential enhancements in dynamic image quality (Heintzmann et al., 2003). The real power of the programmable matrix approach lies in the ability to redefine the array composition, without any additional hardware changes. This enables complete flexibility for optimization of the illumination pattern for the sample and experiments in hand—from low speed high resolution to high speed lower resolution studies.
X. Advantages and Disadvantages of Confocal Microscopy From an experimental point of view, confocal Ca2þ imaging oVers many benefits over conventional fluorescence microscopy, especially with respect to spatial and temporal resolution and the ability to optically section the specimen along the Z-axis down to a resolution of 0.5–1 mm, enabled by the presence of a confocal aperture with a pinhole in the light path that eVectively gives this imaging modality its uniqueness, as well as its name. Importantly, confocal microscopy may be performed in live cells that are electrically, mechanically, and by all other measures, viable. Moreover, confocal microscopy does not impair the ability to manipulate the surrounding environment in terms of solutions in which the cells or specimen are bathed, solution pens, temperature devices, application of pharmacological drugs and inhibitors, patch pipettes, electrical and mechanical manipulators, etc., as long as these do not interfere with the actual light pathway. However, the significant drawbacks with confocal microscopy relate firstly to the unavoidable scattering as well as chromatic aberration with subsequent focal plane oVsetting that reduces confocal signal collection (Bliton et al., 1993) of the illumination light as it penetrates the specimen, especially since wavelengths of 300–600 nm are commonly used for excitation, as those are very prone to scattering; eVectively limiting the penetration depth to 40–50 mm (or less, depending on the laser output and the spectral properties of the specimen), and secondly to the fact that although only the emitted fluorescence produced at or very near to the focal point will be recorded, excitation will still occur along the whole Z-axis of the specimen (given molecules able to be excited are present along the Z-axis). The latter point presents often considerable problems because the photodamage generated along the entire Z-axis may kill live specimens. Though this may be mitigated by lowering the laser power, the restricted light capturing from the small volume accommodated by the pinhole tends to drive toward microscopists increasing laser power. This point is further accentuated by excitation at short wavelengths (300–500 nm) including UV light, as those are severely damaging to live specimens due to the associated high energy. Finally, higher purchase and maintenance costs relative to conventional epifluorescence and widefield microscopy may also represent a disadvantage to the researcher. A diVerent excitation approach with many of the same advantages as confocal laser scanning microscopes as described above, while also substantially limiting the
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major drawbacks of confocal microscopy such as the restricted penetration depth and photodamage, is oVered by multiphoton microscopy systems.
XI. Multiphoton Excitation Laser Scanning Microscopy A conceptually diVerent microscope that also allows optical sectioning and high spatial and temporal resolution live specimen imaging, and that also comes with other added benefits, is the multiphoton microscope. From a biophysical perspective, multiphoton imaging rests on an excitation principle that relies on excitation of the fluorescent molecule by photons that alone do not have enough energy, but need to combine by simultaneously coming in to a very close proximity of the fluorophore. Although the theoretical concept of multiphoton excitation is relatively simple and has been known and also utilized by physicists for many decades, biological applications of multiphoton excitation are of a more recent date (Denk et al., 1990). Whereas in confocal single-photon laser illumination, the fluorophore is excited by the absorption of a single photon, as it provides suYcient energy for the fluorophore to reach an excited state. Upon return to the ground state, a photon of longer wavelength than the excitation photon is emitted, and it is this process that creates the fluorescence. With multiphoton excitation, a fluorophore is excited by the near-simultaneous (within 10 18 s) absorption of two or more photons that combined provide enough energy to promote the fluorescent molecule from a ground state to an excited state; two photons for two-photon (2P) excitation, three photons for three-photon (3P) excitation, etc., with 2P excitation being by far the most common multiphoton excitation modality. Thus, with 2P excitation, the fluorophore absorbs two photons simultaneously, each twice the wavelength and half the energy required for molecular excitation, and likewise, in 3P excitation, each of the three excitation photons have three times the wavelength, but only one-third of the energy compared to singlephoton excitation (Helmchen and Denk, 2005). Once excited, the emitted fluorescence is then proportional to the square of the excitation intensity in 2P absorption (third power in 3P excitation), and this 2P excitation (measured in Goeppert-Meyer Units) occurs only at the focal point, as it is only here that the density of the excitation photons is high enough to ensure a simultaneous photon arrival to the fluorophore that is suYcient to excite it. Though this may not occur with all photons in the volume, the probability of it occurring for the vast majority of the photons is very high. This nonlinear excitation constitutes the most important physical diVerence from confocal single-photon excitation, in that excitation will be confined to a small ellipsoid volume around the focal point, whereas above and below this point, the density of photons suYciently close to one another, or in other words the light intensity, will not be high enough to generate any excitation (Fig. 6). This eVectively means that out-of-focus fluorescence will not be generated, which again removes the need for a confocal aperture with a pinhole in the emission light pathway.
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Fig. 6 Two-photon (2P) excitation microscopy, including a comparison to single-photon confocal microscopy. (A) Schematic of illumination lightbeams, in which excitation occurs along the whole Zaxis with single-photon confocal microscopy, though with highest intensity at the focal point, whereas excitation is confined to a narrow area around the focal point with 2P microscopy. Correspondingly, emission occurs along the entire z-axis with confocal microscopy; though out-of-focus light is blocked by the confocal aperture, whereas emission is confined to a narrow area around the focal point with 2P excitation microscopy. (B) Schematic of the continuous laser used for single-photon excitation and a pulsed titanium:sapphire (Ti:Sapphire) laser used for 2P excitation.
Therefore, the optical sectioning and spatial and temporal resolution one may achieve will be close to that achieved by confocal microscopy or a little poorer, but not better (Cox and Sheppard, 2004). A clear benefit of this is that there need not be a confocal aperture. Although crucial for confocal single-photon microscopy, confocal apertures themselves reduce light transmission, but also require that the emission light path is returned through the scanhead including the mirrors, which further causes a loss of light. Instead, the emitted fluorescence after 2P excitation may be focused directly onto a PMT without having to be descanned, as in confocal microscopy, such that 2P microscope systems are generally more light sensitive. However, the introduction of a confocal aperture with a pinhole into the 2P emission pathway may improve the spatial resolution to a confocal standard.
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Thus, although the capture of the emitted fluorescence may be set up with diVerent light paths in 2P microsscopes compared to confocal single-photon microscopes, it is conceptually similar in that emission light needs to be split by the excitation light with the insertion of an appropriate dichroic mirror. However, in the case of 2P microscopy, the emitted fluorescence will in most cases have a very much shorter wavelength than the excitation wavelength, which is in contrast to confocal single-photon and conventional epifluorescence microscopes. Because of the abovementioned properties of 2P excitation and diVerences to single-photon confocal excitation, 2P excitation oVers several important advantages over single-photon excitation. First, because light scattering declines steeply with increasing wavelengths, red and IR (670–1100 nm) light can penetrate and hence excite fluorescent molecules much deeper into the specimen than singlephoton lasers providing excitation wavelengths in the range from 300 to 600 nm. With suYcient power outputs and optimized optics including corrective optics for pulse dispersion, penetration depths approaching 1000 mm (1 mm) may be achieved, which is considerably deeper than the 40–50 mm achievable with single-photon excitation. Thus, 2P excitation secures an up to 20-fold deeper penetration depths by using excitation light of twice the wavelength. To achieve comparable deep imaging with single-photon confocal microscopy, tissue or layer removal with histologic techniques or penetration by the objective would have been required. Such mechanical approaches would for obvious reasons compromise the ‘‘intactness’’ and viability of the tissue. For this reason, confocal microscopy is mainly restricted to studies of single, isolated cells or tissue surfaces, whereas 2P excitation laser scanning microscopy allows deep tissue imaging of intact organs. Similarly, 2P excitation microscopes are comparably more often upright rather than inverted, to allow for tissue preparation to be set up on the stage, whereupon the objective lens is lowered down to within the optical range for imaging. The opposite is true for confocal microscopes, which more often than not come inverted. Furthermore, even with 2P excitation deep tissue imaging, one can be assured that the vast majority of the fluorescence comes from the focal point and not from residual scattering (scattering is greatly reduced, but not obliterated, by long-wavelength excitation). This is because, even in strongly scattering tissue such as the heart, the density of scattering exciting photons is too low to generate significant fluorescence. Therefore, this together with the long excitation wavelengths also contributes to 2P excitation microscopes being much less sensitive to light scattering than regular widefield epifluorescence or confocal microscopes. However, it should be noted that penetration depths also heavily depend on the specimen, as diVerent tissue properties may degrade the illumination as well as the emitted fluorescent light. In particular, collagen and myelin are known to scatter light and thereby restrict penetration (Helmchen and Denk, 2005). Additionally, IR light is much less phototoxic than shorter wavelengths, such that it correspondingly may cause only negligible photodamage. Also, because of the nonlinear excitation, photodamage and bleaching are restricted to the focal point. Importantly, the reduced photodamage and fluorophore bleaching allows
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for longer imaging periods. 2P excitation spectra of most fluorophores are wider than the equivalent single-photon excitation spectra, whereas the associated emission spectra after 2P and single-photon excitation are not diVerent, such that the same excitation wavelength may potentially excite several fluorophores with distinct emission spectra (Bestvater et al., 2002; Xu et al., 1996). This property will be explored in more detail later in the chapter. Moreover, whereas the positioning of the confocal aperture relative to the light detector as well as the focal plane is critical to ensure that the images of the source and detector apertures are cofocused, this is not the case for 2P excitation microscopes, in which the position of the light detector is not critical, because fluorescence emission will only be generated at and tightly around the focal volume. However, several problems persist with 2P excitation. First, 2P excitation requires very high light intensities; intensities that would instantly vaporize the specimen if the light was delivered continuously. However, this is overcome by using lasers that provide ultrabrief (10 13 s) pulses at very high frequencies ( 80–100 MHz), which thereby generate very high instantaneous energy, but suYciently low average energy to avoid any substantial damage to the specimen. Thus, the distance between each pulse is typically 10 ns, whereas the width of each pulse itself is typically 100 fs (Fig. 6). The high repetition rates match fluorescence lifetimes closely (see above) such that a good balance between excitation eYciency and onset of saturation is achieved (Helmchen and Denk, 2005). The most widely used lasers that fulfill this criterion and provide the necessary wavelengths, are the solid state titanium:sapphire (Ti:Sapphire) oscillating (pulsed) lasers. These lasers are tunable, such that the latest versions are capable of delivering wavelengths within the range 670– 1100 nm, thus, from visible red to infrared (IR), though this range is continuously expanding while also coming with higher power outputs, as manufacturers keep developing their product lines. Though most 2P excitation applications may not be power-limited, more available power does benefit applications requiring deep tissue penetration. Typical power outputs in the latest Ti:Sapphire lasers may exceed 4 W at 800 nm, though the optics cause a major loss in power from the outlet of the laser to the focal point of the objective lens. Glass components of the optical pathway also cause a dispersion of the 100 fs laser pulses, but this may be compensated for by corrective optics in order to maximize 2P excitation (Diels et al., 1985). Other limitations of 2P excitation microscopy are that reflected light imaging is not possible; only fluorescence imaging, and that it is not suitable for imaging highly pigmented specimens, as these absorb IR and near-IR light.
XII. Ca2þ Indicators for Use in Confocal and Multiphoton Microscopy For many years, biology has benefited from the fluorescent dyes or constructs that bind and therefore can measure the free concentration of Ca2þ [Ca2þ]. These indicators have been used to examine the levels and time course of changes
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in [Ca2þ] within the cytosol and the various organelles within the cell (e.g., nucleus, mitochondria, sarco/endoplasmic reticulum). The two main categories of molecules used for this purpose are small synthetic organic molecules based on the fast Ca2þ buVer BAPTA (Tsien, 1980) or modified versions of natural Ca2þ binding proteins (Miyawaki et al., 2003). Both categories ‘‘sense’’ Ca2þ by chelating the ion which changes the structure/chemical properties of the ligands. Several modes of fluorescence have been utilized to report Ca2þ (summarized in Fig. 7). Absorbance/quantum yield: In this case, Ca2þ binding causes a change in the intensity of the fluorescence from the dye; typically, Ca2þ binding causes an increase in fluorescence (Fig. 7A). This mode is the most common employed by the fluorescent Ca2þ indicators used in confocal or 2P microscopy. In particular,
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Fluo-3, Fluo-4, and Oregon Green all show dramatic increase in fluorescence upon binding to Ca2þ, while Fura-Red fluorescence decreases upon Ca2þ binding. The optical arrangement required for these dyes is the simplest, that is, single excitation and emission wavelengths. Spectral shift: In this case, Ca2þ binding causes a shift in either the excitation or emission wavelengths. Fura-2 and related dyes show a shift in the excitation spectrum of the dye with minimal changes in the emission (Fig. 7B). In contrast, Indo-1 and related dyes show large shifts in the emission spectrum of the dye with minimal changes in the excitation spectrum. This spectral shift is used to allow ratiometric measurements. In the case of Fura-based dyes, while exciting at 340 nm, a rise of Ca2þ will cause an increase in fluorescence, whereas while exciting at 380 nm causes a decrease. The ratio of fluorescence at these two wavelengths is a unique function of the [Ca2þ] and is independent of the concentration of the dye. This is particularly useful when measuring cells that move and thereby alter the amount of dye within the light path, or comparing cell compartments with diVerent dye concentrations (e.g., nucleus versus the cytosol). However, despite all these advantages, the Fura- and Indo-based dyes are rarely used in confocal microscopy because excitation wavelengths are short (< 400 nm) and therefore not convenient for commonly available lasers. The shortest wavelength routinely available in commercial systems is 405 nm, which can be used to excite Fura-based dyes close to the 380 nm excitation maximal. In this mode, Fura acts as an inverse indicator, a property that has some value in single-photon confocal and 2P imaging (Ogden et al., 1995; Wokosin et al., 2004). Fluorescence lifetime: In this case, the decay of fluorescence at the end of a pulse of excitation light will take a finite time (ns) to decay. The time course of decay is aVected by the binding of Ca2þ (Fig. 7C). In the case of Fluo-3, the decay of fluorescence or lifetime is shorter for the Ca2þ bound form (Sanders et al., 1995). Since the lifetime is independent of the concentration of the dye, the relative population of bound and free dye can easily be calculated from this technique. However, the nontrivial analysis of emission decay required to obtain lifetime data has minimized the use of this technique. FRET eYciency: In this case, a donor molecule absorbs photons and can transfer the associated energy to a close by acceptor molecule via a nonradiative process that operates at distances less than the wavelength of light (up to 10 nm). The closely adjacent molecule accepts the energy transfer (thus excites) and then emits light at a distinct wavelength (Fig. 7D). The most popular pairs of donor and acceptor molecules are cyan fluorescent proteins (donor) and yellow fluorescent proteins (acceptor). The eYciency of the energy transfer depends on the proximity of the two molecules; within 10 nm, the eYciency is high but this drops dramatically as the distance between the two molecules increases (proportional to 1/(distance)6). This mechanism can be used to detect Ca2þ binding to either construct since the change in tertiary structure will alter FRET eYciency and therefore altered FRET signal.
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XIII. Use of Dyes for Single-Photon Confocal Microscopy In theory, all the fluorescent Ca2þ indicators created for cell biology can be used in confocal or 2P excitation modes, although in practice, only a limited number of dyes are routinely used for a series of practical considerations. By far, the majority of confocal applications use single-wavelength excitation and emission dyes. These dyes have several advantages: (i) their excitation wavelength is 500 nm or longer and therefore can use the emission from readily available Argon or KryptonArgon lasers, and (ii) the majority of dyes have a good dynamic range (see below). But they also suVer from a series of disadvantages: (i) calibration is diYcult because the signal will be a function of both the concentration of the dye (unknown and variable) and the concentration of Ca2þ and (ii) dye signal may vary with time due to loss of the dye from the volume or due to photobleaching. All dyes photobleach, but some dyes are more susceptible than others; in particular, Fluo-3 and Fluo-4 are amongst those that most rapidly bleach (Thomas et al., 2000). Dyes that show significant spectral shifts can be used in a single wavelength mode, for example, Fura-based dyes can be imaged in confocal microscopy by using a 405 nm laser. This produces an inverse reporter since fluorescence decreases as Ca2þ increases (Wokosin et al., 2004). Similar measurements using Indo-based dyes are less easy because the narrow excitation spectrum means that 405 nm laser light only poorly excites the dye and Indo-based dyes appear to be inherently more prone to photobleaching.
XIV. Use of Dyes for 2P Excitation Microscopy The utility of a dye for imaging Ca2þ using 2P excitation does not follow directly from the behavior of the dye in single-photon excitation. There is no guarantee that 2P excitation will successfully excite the dye since the eYciency of two-photon excitation appears to be relative to the fluorophore structure (Kim et al., 2008). A number of Ca-sensitive indicators have been studied over a limited range of 2P wavelengths (Xu et al., 1996), some dyes (e.g., Oregon Green) have a better 2P cross-section than the more common single wavelength dyes (Fluo-3); none showed the expected increase in cross-section as the excitation wavelength approached 900 nm. In contrast, the 2P cross-section of Fura-based dyes appeared readily excitable by wavelengths approximately to double the appropriate singlephoton wavelength (Wokosin et al., 2004). In particular, 800 nm light provides an inverse Ca2þ-sensitive signal that corresponds to the excitation of this dye at 400 nm, as advocated previously (Ogden et al., 1995). However, few studies to date have explored the more commonly used dyes (Fluo-3/4 and Rhod-2) at the longer wavelengths achievable currently with tunable Ti:Sapphire lasers (up to 1100 nm). As shown in Fig. 8, the Fluo-based dyes do not show the peak of fluorescence anticipated from approximately double single-photon wavelengths ( 1000 nm). The small peaks in the excitation spectrum observed at 400–470 nm
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during single-photon excitation were also paralleled by similar spectra at 800–950 nm during 2P excitation, but longer wavelengths failed to produce the increased fluorescence anticipated from the single-photon spectrum. The behavior of the Rhod-dyes (related structurally to Fluo-dyes) has yet to be examined in detail mainly because accessing wavelengths > 1100 nm is technically diYcult. The practical application of 2P excitation to excite Ca2þ-sensitive dyes is limited; the majority of applications used 800 nm to excite either Fluo-3 or Rhod-2. In doing so, these studies are using the higher powers available from lasers at these wavelengths, rather than attempting to access the higher quantum yields that may be present at longer wavelengths. Further work is clearly required to determine the optimal 2P conditions to excite these readily available dyes at 1000– 1100 nm, or alternatively design new dyes with fluorophore structures that are more easily excited by the 2P approach (Kim et al., 2008).
XV. Is It Worth Converting the Intracellular Fluorescence Signal to [Ca2þ]? Ca2þ-sensitive dyes are frequently used in conjunction with confocal microscopy to simply indicate the timing or frequency of transient Ca2þ events. Under these circumstances, it is not considered necessary to convert the fluorescence signal into estimates of intracellular [Ca2þ]. This is avoided partly for experimental ease, since conversion requires knowledge of the concentration of the dye and its aYnity for Ca2þ, and partly because absolute Ca2þ concentrations are not considered vital to the interpretation of the experiments. In a number of cases this may be justified, but whenever amplitude or time course of a Ca2þ transient is considered an important variable, then calibration becomes essential for the following several reasons: 1. It is important to distinguish changes in background Ca2þ from one experimental scenario to the next since this determines the subsequent intracellular Ca2þ buVer power. Any event that generates a Ca2þ transient, for example, Ca2þ influx via plasmalemmal Ca2þ channels or Ca2þ release from an internal store, will increase total intracellular Ca2þ by a specific amount, but the extent to which this increases the free cytosolic Ca2þ concentration will depend on the cellular buVer power for Ca2þ. This is illustrated in Fig. 9, where total cellular Ca2þ is increased by a standard amount; the subsequent free Ca2þ transient amplitude can be 40% larger simply because of small ( 10%) changes in resting [Ca2þ]. Therefore, if the basal Ca2þ concentration changes, the interpretation of changes in transient amplitude has to be made with caution. 2. Intracellular [Ca2þ] levels that almost saturate the indicator cannot be used to examine moderate changes in the amplitude of the Ca2þ transient. In the absence of information concerning the maximal Ca2þ signal, it is diYcult to know how close the dye is to saturation and therefore how sensitive the signal is to changes in peak Ca2þ level.
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XVI. Calibration of Single Wavelength Dyes Calibration of single wavelength dyes is based on two main assumptions: firstly, that the dye is at equilibrium with intracellular Ca2þ, that is, the kinetics of the change of intracellular Ca2þ are slow compared to the rate constants for association and dissociation. For the commonly used Fluo-type dyes at an intracellular concentration of < 100 mM, the half time for association and dissociation are of the order of 1 ms and therefore for most circumstances the equilibration assumption holds. The second assumption is that all the dye molecules sense the same amount of light, that is, there is no filtering intrinsic to the biological preparation or by the dye itself. Typically, the absorption coeYcient of Ca2þ indicators is of the order of 50,000 M 1 cm 1, therefore significant absorbance (> 0.05) would occur in a cell 10 mm thick containing > 100 mM dye. This consideration is important for singlephoton excitation and is one of the constraints that limit single-photon imaging to thin (< 50 mm) specimens.
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With these two criteria satisfied, the Ca2þ concentration can be calculated from the fluorescence from single wavelength dyes using the following equation: 2þ ¼ Kd ðF Fmin Þ=ðFmax F Þ Ca where F is the fluorescence signal from the cell/tissue; Kd the dissociation constant of Ca2þ for the indicator (units M); Fmin the minimum fluorescence achieved when the dye is essentially Ca2þ free, which practically can be approximated by exposing the dye to a [Ca2þ], that is, 0.01Kd of the dye; and Fmax is the fluorescence achieved when the dye is completely Ca2þ bound, which practically can be achieved with [Ca2þ] of 100Kd of the dye. Measurements of these constants with some degree of precision inside a cell are, however, diYcult. The dissociation constant can be measured outside the cell in solutions approximating the intracellular mileau, but it has been a frequent observation that the value of the Kd is altered by the intracellular environment in a way that is diYcult to mimic by solution chemistry, for example, mimicking intracellular viscosity and the range of negatively charged intracellular proteins (Poenie, 1990). Therefore, the best practice is to measure the dissociation constant within the cell type of interest. The easiest way to achieve this is by using a glass microelectrode to gain access to the intracellular space. The use of a series of solutions with a high concentration of Ca2þ buVer (EGTA or BAPTA) with specific [Ca2þ] can be used to make a series of single cell measurements to allow estimation of Kd. However, it is important to note that this technique cannot be applied to multicellular preparations where multiple cells in a tissue are diVerentially loaded with the dye.
XVII. Estimation of Fmax Values This should be measured on a cell-to-cell basis even within multicellular preparations and involves exposing the inside of the cell to 50 mM or higher Ca2þ, depending on the aYnity of the dye for Ca2þ. These levels are generally toxic to cells, but if tolerated for a short time (1–2 s), this may be suYcient to estimate Fmax. These intracellular [Ca2þ] levels can be achieved rapidly within single cells by perfusion with a Ca2þ ionophore and raised extracellular Ca2þ (Loughrey et al., 2003). A second ingenious method used in single voltage clamp experiments is to use an amphotericin-containing patch pipette that facilitates monovalent cation exchange across the membrane within the patch and therefore allows low resistance access to the cell. At the end of the experiment, the membrane is ruptured under the patch using a rapid pressure step and the resultant influx of Ca2þ from the patch pipette generates a rapid rise of intracellular [Ca2þ] that can be used to assess Fmax (Diaz et al., 2001). A simpler but less reliable method is to simply use the microelectrode to penetrate the cell and allow extracellular Ca2þ influx in order to record Fmax, but generally Ca2þ influx occurs in parallel with a rapid loss of intracellular dye so the signals would have to be interpreted with caution.
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Depending on the tissue, there are alternative approaches to estimate Fmax; in some nerve cells, rapid frequent stimulation of cells can generate intracellular Ca2þ levels that approach saturation of the dye (Maravall et al., 2000), thus allowing Fmax values to be estimated.
XVIII. Estimation of Fmin or the Dynamic Range of the Dye Estimation of Fmin or the dynamic range of the dye is more diYcult. The ratio of Fmax/Fmin measured outside the cell cannot be assumed to apply inside; estimates suggesting that values of 70–80% of those seen in free solutions are common (Poenie, 1990). Again, an averaged value can be obtained using patch pipettes as a means of establishing buVered [Ca2þ] inside cells. Alternatively, the Fmin in each experiment can be estimated if the intracellular Ca2þ can be lowered to 1–10 nM (for Fluo-3) by decreasing extracellular Ca2þ, but this assumes that intracellular Ca2þ can be readily manipulated by changes in the extracellular environment which is not always the case for every cell type. The eVect of under- or overestimation of the dynamic range of the indicator is shown in Fig. 10C. Based on the reported dynamic range of Fluo-based dyes ( 100), large under/overestimates of the dynamic range (by up to 30%) cause only small errors in Ca2þ estimation and only in the lower range of [Ca2þ] values relative to the Kd of the dye.
XIX. Consequence of Errors in Estimation of Intrinsic and Dye Fluorescence Prior to conversion of the indicator-based fluorescence to [Ca2þ], the background or intrinsic fluorescence of the cell/tissue has to be subtracted from the signal. All cells have an intrinsic fluorescence mainly due to the metabolites beta nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD); their excitation wavelengths are 350–500 nm and emission wavelengths 450–600 nm. The relative fluorescence of these two metabolites depends on the metabolic state of the cell/tissue and degree of photobleaching. Thus, intrinsic cellular fluorescence is significant and variable. The most advisable approach is to use a dye with a significant basal fluorescence that is many times (> 10) that of the intrinsic value. This cannot always be achieved; the Fluo-based and Rhodaminebased dyes are by far the most popular dye groups used in confocal and 2P excitation microscopy. Their main attraction is a large dynamic range as a result of a low fluorescence signal from the Ca2þ free form. In this situation, Fmin values are frequently comparable to that of the intrinsic fluorescence of the cell and therefore it is important to quantify either by parallel measurements on nonloaded tissue or from a single cell prior to the introduction of the dye. Error in estimation of background fluorescence (which can be up to 100%) has dramatic eVects on the calculation of
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intracellular [Ca2þ], particularly at either end of the sensitive range of the indicator, as shown in Fig. 10A. If the dye has a Ca2þ aYnity midway between the extremes of intracellular Ca2þ, then the error can be small and approximately constant. But if the dye has a lower Ca2þ aYnity, which may be desirable to resolve changes in peak Ca2þ, then errors associated with the minimum cellular Ca2þ levels can be large.
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These errors can be compounded by errors in estimates of fluorescence or the variability of signals from one cell to the next. Fig. 10B shows the errors in Ca2þ based on simple errors in fluorescence changes. The graph illustrates the risk inherent in using dyes with a relatively high aYnity relative to the physiological signal. Small errors in the range of fluorescence signals translate to large errors of intracellular Ca2þ such that the ability to discriminate changes in maximum physiological response is severely impaired. This can be significantly improved by using lower aYnity dyes, but at the cost of poor resolution of minimum or background intracellular [Ca2þ].
XX. Multimodal and Multiple Fluorophore Confocal and Multiphoton Microscopy Although Ca2þ is an important signaling molecule in a variety of cell types, it by no means operates alone. Rather, Ca2þ both temporally and spatially interacts with many other properties and processes in the cell that only in concert orchestrate cellular function. Thus, some of these processes are dictated by Ca2þ, but some or not. A good example of this interplay is excitation–contraction coupling in muscle cells, in which the action potential depolarizes the plasma membrane of the cell, which causes a small influx of Ca2þ through the membrane. This inward Ca2þ current stimulates the ryanodine receptor to release bulk Ca2þ from the sarcoplasmic reticulum, which upon binding to the myofilaments induces the actin–myosin interaction and the subsequent cellular contraction (Bers, 2002). The cellular contraction may be imaged by simple black-and-white contrast edge-detection microscopy, but this is not the case for the intracellular Ca2þ and membrane potential characteristics that both require more sophisticated methods such as fluorescence microscopy. Thus, simultaneous imaging with the use of multiple fluorophores present at the same time in the specimen or combinations of diVerent imaging modalities in some sense is required for capturing complex information. Thus, loading or injecting the specimen with multiple fluorophores allows for simultaneous recording of diVerent signals, or if simultaneous recordings are not technically possible, diVerent signals may be recorded sequentially without having to manipulate, move, or in any other way perturb the specimen between recordings. In the latter case, only the optical pathways of the microscope would be altered between recordings, whereas the specimen would not, since it would already be loaded with diVerent fluorophores. The use of multiple fluorophores require either the ability to direct separate emission wavelength bands onto diVerent light detectors, or to spectrally separate diVerent fluorophores by diVerent excitation wavelengths. Depending on the hardware, both confocal and multiphoton microscopes can fulfill these requirements and therefore allow for measurements with multiple fluorophores. Such experiments can be done by simultaneously loading the
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specimen with a Ca2þ-sensitive fluorophore and a fluorophore that is sensitive for another characteristic of the cell, which may also be Ca2þ in a diVerent compartment of the cell with diVerent dynamics or a diVerent concentration range, or a second fluorophore that may be sensitive to the plasma membrane voltage in an excitable cell (also called a potentiometric dye). Measurements of Ca2þ and membrane voltage (resting membrane potentials and action potentials) may then be conducted either simultaneously by exciting both fluorophores at the same time and capture spectrally diVerent fluorescence emission signals, or sequentially by exciting each fluorophore separately, that is, one after the other, under otherwise similar experimental conditions. The latter approach would assume that the experimental conditions remain the same. Several factors may necessitate this, such as an inability to diVerentiate between diVerent emission signals, or an inability to excite more than one fluorophore at any given time, for example, if the excitation spectra do not overlap and only one excitation wavelength may be delivered at one time. The advantage of using multiple fluorophores either simultaneously or sequentially is to increase the information content of the imaging, especially how diVerent processes relate to each other spatially and temporally. However, several issues may limit the applicability of such measurements. Introducing a fluorophore to the specimen may also change the dynamics of the cellular parameter of interest, especially in live specimens that rely on stable and constant intra- and extracellular environments. For instance, most Ca2þ indicators are also Ca2þ chelators that buVer free Ca2þ, and most fluorophores or the medium they are delivered in may change biochemical and biophysical properties of the intracellular environment. This may be accentuated by simultaneous loading with several dyes. DiVerent dyes may also quench, sequester, or in other ways inhibit each other. Finally, excitation in itself may cause changes or damage to the specimen, and although this to some degree is unavoidable, the degree of change or damage may be diVerent or even accentuated during sequential recordings. The single-photon excitation and emission spectra of multiple Ca2þ- and voltage-sensitive fluorescent dyes are well known. Clearly, some dyes have overlapping excitation or emission peaks, or present with broad excitation or emission spectra such that even if the peaks are separated from one another, the tails of the spectra still overlap considerably. Overlapping excitation spectra means that diVerent dyes may be excited simultaneously, but overlapping emission spectra may result in severely reduced signal specificity, and therefore, certain combinations of fluorescent dyes may be less applicable, such as the potentiometric Di-4ANEPPS and Di-8-ANEPPS dyes, and the Ca2þ-sensitive Fluo-3 dye, all extensively used by numerous laboratories for single-fluorophore purposes. All of these dyes have single-photon excitation peaks at 480–500 nm and emission peaks at 520–610 nm, respectively, with especially Di-4-ANEPPS and Di-8-ANEPPS having very broad emission spectra that peak at 610 nm, but that considerably overlap with the Fluo-3 emission spectrum, even though the latter has its peak at 525 nm and therefore numerically diVers from Di-4-ANEPPS and Di-8-ANEPPS and is more narrow (Fig. 11A). This problem may to some degree
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be avoided as the Ca2þ and voltage signals are spatially separated between intracellular and membrane compartments of the cell, though in muscle cells this may turn out to be diYcult because of the dense network of plasma membrane transverse tubules that penetrate into the interior of the cell. The same overlap problem exists with the voltage-sensitive RH-237 and the Ca2þ-sensitive Rhod-2 dyes, with emission peaks occurring at 580 and 660 nm, respectively, but with especially the RH-237 emission spectrum being very broad (Fig. 11B). In contrast, Fluo-3 and RH-237 are more distinctly separated from one another. Both Fluo-3 and RH-237 dyes may be excited by the same single-photon excitation wavelength at
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500 nm (though RH-237 would not be optimally excited by this wavelength), but have emission spectra that may be spectrally diVerentiated, as Fluo-3 has a narrow emission spectrum that peaks at 525 nm, whereas RH-237 peaks at 660 nm with a broad spectrum (Fig. 11C). Thus, this combination appears attractive as it diVerentiates the Ca2þ and membrane potential signals, for which it has also been used successfully (Fast and Ideker, 2000). However, several problems arise when transferring from single-photon to 2P excitation, although several of the voltage-sensitive dyes present with consistent and reproducible 2P excitation spectra that very much resemble the double singlephoton excitation spectra and may thus be confidently used for meaningful multiphoton imaging. In contrast, the 2P behavior of many of the Ca2þ-sensitive dyes is somewhat diYcult to interpret (see previous discussion and Fig. 8), although the ratiometric Fura dyes may be 2P excited in order to provide a meaningful Ca2þ signal that also captures transient changes over a millisecond scale with high fidelity (Wokosin et al., 2004). This may be because the Fura dyes have a singlephoton excitation spectrum in the UV range (340–380 nm), and therefore the 2P excitation spectrum, which approximately is double the single-photon spectrum, occurs at 800 nm wavelengths, in which the 2P laser power outputs are not limited. Moreover, this study indicated that several of the Fura dyes, in particular Fura-4F, may work well when excited with a single IR wavelength, despite their use as ratiometric single-photon dyes, as judged by the dynamic ranges and SNR obtained during 2P excitation microscopy in single cardiac muscle cells during diVerent Ca2þ conditions. In contrast, the Fluo- and Rhod-based Ca2þ-sensitive dyes, all with single-photon excitation peaks at 500–550 nm, present with 2P excitation spectra that are not immediately predicted by the doubled single-photon spectra (Fig. 8). In these cases, the 2P excitation spectra are at least partly broken up and appear blueshifted compared to the doubled single-photon spectra. Although doubling the single-photon excitation spectrum is often a good predictor for the 2P excitation spectrum, deviations from this do occur, although these deviations may neither be systematic nor well understood (Xu et al., 1996; Zipfel et al., 2003). Alongside this, a reoccurring problem is that the available Ti:Sapphire pulsed 2P lasers are power-limited at the long wavelengths of 1000–1100 nm that would correspond to the doubled single-photon excitation spectra of Fluo-3 and Rhod-2. Because not all fluorophores are easily transferable from single-photon excitation, for which they were developed, to 2P excitation, this therefore has made it problematic to use multiple fluorophores simultaneously during 2P excitation microscopy, and the issue has not yet been fully resolved. A diVerent approach to capture more complex information has been to combine several multimodal microscopy techniques in ways that also encompass confocal and multiphoton systems, but also this comes with both advantages and disadvantages. For instance, diVerent modes of contrast used on the same specimen may increase the information extracted from the images and reduce artifacts. Multimodal microscopy may also allow for a wider repertoire of fluorophores. However, if confocal and multiphoton imaging are combined, it requires descanning and insertion of a
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confocal aperture with a pinhole into the light pathway, which may reduce the fluorescence capture after 2P excitation and thus lead to a loss of signal, though a confocal aperture may also be set up to increase the spatial resolution of multiphoton images, by restricting the PSF tails. Because of these limitations, the reality is often that it is diYcult, though not impossible, to achieve optimal performance from each individual mode when several modes are combined. Nonetheless, the advantages of simultaneous or near-simultaneous light capture by diVerent modes of microscopy may, under the right circumstances, far outweigh the disadvantages. Examples include combinations of confocal or multiphoton with epifluorescence or diVerential interference contrast microscopes to capture light emission restricted to the focal plane as well as capturing a widefield view, either simultaneously or sequentially without having to reorient or replace the specimen. Other options also include setting up a microscope system that combines confocal and 2P excitation imaging modes, or 2P excitation and second-harmonic generation (SHG) imaging. Although these applications tend to serve narrow and specific purposes, they may allow for imaging of local versus global Ca2þ signaling, or Ca2þ signaling in combination with for example, metabolic parameters by using 2P excitation to excite metabolites such as NADH and FAD, or collagen that in particular contributes to the SHG signal (Masters, 2006). A final example of multimodal microscopy techniques that may successfully be combined includes the combination of FRET and FLIM imaging to quantify FRET between two fluorophores, as in the case of the Ca2þ-sensitive cameleon described above. These examples are not exhaustive, but serve to illustrate the potential of combining diVerent fluorophores or microscopy modalities in order to gain information of a more detailed nature. References Bers, D. M. (2002). Cardiac excitation–contraction coupling. Nature 90, 182–189. Bestvater, F., Spiess, E., Stobrawa, G., Hacker, M., Feurer, T., Porwol, T., Berchner-Pfannschmidt, U., Wotzlaw, C., and Acker, H. (2002). Two-photon fluorescence absorption and emission spectra of dyes relevant for cell imaging. J. Microsc. 208, 108–115. Bliton, C., Lechleiter, J., and Clapham, D. E. (1993). Optical modifications enabling simultaneous confocal imaging with dyes excited by ultra-violet and visible wavelength light. J. Microsc. 169, 15–26. Bootman, M. D., Collins, T. J., Peppiatt, C. M., Prothero, L. S., MacKenzie, L., De Smet, P., Travers, M., Tovey, S. C., Seo, J. T., Berridge, M. J., Ciccolini, F., and Lipp, P. (2001). Calcium signalling—An overview. Semin. Cell Dev. Biol. 12, 3–10. Bullen, A. (2008). Microscopic imaging techniques for drug discovery. Nat. Rev. Drug Discov. 7, 54–67. Callamaras, N., and Parker, I. (1999). Construction of a confocal microscope for real-time x–y and x–z imaging. Cell Calcium 26, 271–279. Cleemann, L., Di Massa, G., and Morad, M. (1997). Ca2þ sparks within 200 nm of the sarcolemma of rat ventricular cells: Evidence from total internal reflection fluorescence microscopy. Adv. Exp. Med. Biol. 430, 57–65. Conchello, J. A., and Lichtman, J. W. (2005). Optical sectioning microscopy. Nat. Methods 2, 920–931. Conchello, J. A., Kim, J. J., and Hansen, E. W. (1994). Enhanced 3-D reconstruction from confocal scanning microscope images. II: Depth discrimination vs. signal-to-noise ratio in partially confocal images. Appl. Opt. 33, 3740–3750.
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Godfrey Smith et al. Cox, G., and Sheppard, C. J. R. (2004). Practical limits of resolution in confocal and non-linear microscopy. Microsc. Res. Tech. 63, 18–22. Denk, W., Strickler, J., and Webb, W. (1990). Two-photon laser scanning fluorescence microscopy. Science 248, 73–76. Diaz, M. E., TraVord, A. W., and Eisner, D. A. (2001). The eVects of exogenous calcium buVers on the systolic calcium transient in rat ventricular myocytes. Biophys. J. 80, 1915–1925. Diels, J. C., Fontaine, J. J., McMichael, I. C., and Simoni, F. (1985). Control and measurement of ultrashort pulse shapes (in amplitude and phase) with femtosecond accuracy. Appl. Opt. 24, 1270–1282. Fast, V., and Ideker, R. (2000). Simultaneous optical mapping of transmembrane potential and intracellular calcium in myocyte cultures. J. Cardiovasc. Electrophysiol. 11, 547–556. Hanley, Q. S., Verveer, P. J., Gemkow, M. J., Arndt-Jovin, D., and Jovin, T. M. (1999). An optical sectioning programmable array microscope implemented with a digital micromirror device. J. Microsc. 196, 317–331. Heintzmann, R., Sarafis, V., Munroe, P., Nailon, J., Hanley, Q. S., and Jovin, T. M. (2003). Resolution enhancement by subtraction of confocal signals taken at diVerent pinhole sizes. Micron 34, 293–300. Helmchen, F., and Denk, W. (2005). Deep tissue two-photon microscopy. Nat. Methods 2, 932–940. Jares-Erijman, E. A., and Jovin, T. M. (2003). FRET imaging. Nat. Biotechnol. 21, 1387–1395. Kettlewell, S., Cabrero, P., Nicklin, S. A., Dow, J. A. T., Davies, S., and Smith, G. L. (2009). Changes of intra-mitochondrial Ca2þ in adult ventricular cardiomyocytes examined using a novel fluorescent Ca2þ indicator targeted to mitochondria. J. Mol. Cell. Cardiol. 46, 891–901. Kim, H. M., Kim, B. R., An, M. J., Hong, J. H., Lee, K. J., and Cho, B. R. (2008). Two-photon fluorescent probes for long-term imaging of calcium waves in live tissue. Chemistry 14, 2075–2083. Levitt, J. A., Matthews, D. R., Ameer-Beg, S. M., and Suhling, K. (2009). Fluorescence lifetime and polarization-resolved imaging in cell biology. Curr. Opin. Biotechnol. 20, 28–36. Lichtman, J. W., and Conchello, J. A. (2005). Fluorescence microscopy. Nat. Methods 2, 910–919. Loughrey, C. M., MacEachern, K. E., Cooper, J., and Smith, G. L. (2003). Measurement of the dissociation constant of Fluo-3 for Ca2þ in isolated rabbit cardiomyocytes using Ca2þ wave characteristics. Cell Calcium 34, 1–9. Maravall, M., Mainen, Z. F., Sabatini, B. L., and Svoboda, K. (2000). Estimating intracellular calcium concentrations and buVering without wavelength ratioing. Biophys. J. 78, 2655–2667. Mashanov, G. I., Tacon, D., Knight, A. E., Peckham, M., and Molloy, J. E. (2003). Visualizing single molecules inside living cells using total internal reflection fluorescence microscopy. Methods 29, 142–152. Masters, B. R. (2006). ‘‘Confocal Microscopy and Multiphoton Excitation Microscopy. The Genesis of Live Cell Imaging.’’ The International Society for Optical Engineering Press, Bellingham, WA. Miyawaki, A., Llopis, J., Heim, R., McCaVery, J. M., Adams, J. A., Ikura, M., and Tsien, R. Y. (1997). Fluorescent indicators for Ca2þ based on green fluorescent proteins and calmodulin. Nature 388, 882–887. Miyawaki, A., Mizuno, H., Nagai, T., and Sawano, A. (2003). Development of genetically encoded fluorescent indicators for calcium. Methods Enzymol. 360, 202–225. Nagai, T., Sawano, A., Park, E. S., and Miyawaki, A. (2001). Circularly permuted green fluorescent proteins engineered to sense Ca2þ. Proc. Natl. Acad. Sci. USA 98, 3197–3202. Ogden, D. (ed.) (1994). Microelectrode Techniques. ‘‘The Plymouth Workshop Handbook.’’ 2nd edn. The Company of Biologists Ltd, Cambridge, UK. Ogden, D., Khodakhah, K., Carter, T., Thomas, M., and Capiod, T. (1995). Analogue computation of transient changes of intracellular free Ca2þ concentration with the low aYnity Ca2þ indicator furaptra during whole-cell patch-clamp recording. Pflugers Arch. 429, 587–591. Poenie, M. (1990). Alteration of intracellular Fura-2 fluorescence by viscosity: A simple correction. Cell Calcium 11, 85–91. Sanders, R., Draaijer, A., Gerritsen, H. C., Houpt, P. M., and Levine, Y. K. (1995). Quantitative pH imaging in cells using confocal fluorescence lifetime imaging microscopy. Anal. Biochem. 227, 302–308.
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Tanaami, T., Otsuki, S., Tomosada, N., Kosugi, Y., Shimizu, M., and Ishida, H. (2002). High-speed 1-frame/ms scanning confocal microscope with a microlens and Nipkow disk. Appl. Opt. 41, 4704–4708. Thomas, D., Tovey, S., Collins, T. J., Bootman, M. D., Berridge, M. J., and Lipp, P. (2000). A comparison of fluorescent Ca2þ indicator properties and their use in measuring elementary and global Ca2þ signals. Cell Calcium 28, 213–223. Tsien, R. Y. (1980). New calcium indicators and buVers with high selectivity against magnesium and protons: Design, synthesis, and properties of prototype structures. Biochemistry 19, 2396–2404. Tsien, R. Y., and Bacskai, B. J. (1995). Video-rate confocal microscopy. In ‘‘Handbook of Biological Confocal Microscopy.’’ (J. B. Pawley, ed.), Plenum Press, New York. Wang, E., Babbey, C. M., and Dunn, K. W. (2005). Performance comparison between the high-speed Yokogawa spinning disc confocal system and single-point scanning confocal systems. J. Microsc. 218, 148–159. Webb, R. H. (1999). Theoretical basis of confocal microscopy. Methods Enzymol. 307, 3–20. Willig, K. I., Rizzoli, S. O., Westphal, V., Jahn, R., and Hell, S. W. (2006). STED microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosis. Nature 440, 935–939. Wilson, T., Juskaitis, R., Neil, M. A., and Kozubek, M. (1996). Confocal microscopy by aperture correlation. Opt. Lett. 21, 1879–1881. Wokosin, D. L., Loughrey, C. M., and Smith, G. L. (2004). Characterization of a range of Fura dyes with two-photon excitation. Biophys. J. 86, 1726–1738. Xu, C., Zipfel, W. R., Shear, J. B., Williams, R. M., and Webb, W. W. (1996). Multiphoton fluorescence excitation: New spectral windows for biological nonlinear microscopy. Proc. Natl. Acad. Sci. USA 93, 10763–10768. Zipfel, W. R., Williams, R. M., and Webb, W. W. (2003). Nonlinear magic: Multiphoton microscopy in the biosciences. Nat. Biotechnol. 21, 1369–1377.
CHAPTER 10
The Use of Aequorins to Record and Visualize Ca2þ Dynamics: From Subcellular Microdomains to Whole Organisms Sarah E. Webb,* Kelly L. Rogers,† Eric Karplus,‡ and Andrew L. Miller* *Biochemistry and Cell Biology Section and State Key Laboratory of Molecular Neuroscience Division of Life Science HKUST, Clear Water Bay Kowloon, Hong Kong, PR China †
The Walter and Eliza Hall Institute of Medical Research Parkville, Australia
‡
Science Wares Inc. Falmouth Massachusetts, USA
Abstract I. Introduction II. Expression of Apoaequorin, GFP-Apoaequorin, and Other Apoaequorin-Based Spectral Variants in Cells, Tissues, and Whole Organisms A. Expression of Apoaequorin B. Expression of GFP-Apoaequorin C. Summary of Section II III. Introducing Coelenterazines into Cells, Tissues and Embryos IV. Techniques for Detecting Aequorin Luminescence V. Conclusions References
Abstract In this chapter, we describe the practical aspects of measuring [Ca2þ] transients that are generated in a particular cytoplasmic domain, or within a specific organelle or its periorganellar environment, using bioluminescent, METHODS IN CELL BIOLOGY, VOL. 99 Copyright 2010, Elsevier Inc. All rights reserved.
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genetically encoded and targeted Ca2þ reporters, especially those based on apoaequorin. We also list examples of the organisms, tissues, and cells that have been transfected with apoaequorin or an apoaequorin-BRET complex, as well as of the organelles and subcellular domains that have been specifically targeted with these bioluminescent Ca2þ reporters. In addition, we summarize the various techniques used to load the apoaequorin cofactor, coelenterazine, and its analogs into cells, tissues, and intact organisms, and we describe recent advances in the detection and imaging technologies that are currently being used to measure and visualize the luminescence generated by the aequorinCa2þ reaction within these various cytoplasmic domains and subcellular compartments.
I. Introduction One of the most significant recent developments in the Ca2þ signaling field has been the general acceptance of the wide-spread heterogeneity of Ca2þ activity within individual cells; not only at rest, but also most importantly, during stimulation (Berridge, 2009; Rizzuto and Pozzan, 2006; Rutter et al., 2006; Whitaker, 2008). This has led to the concept of dynamic subcellular ‘‘Ca2þ microdomains.’’ As suggested by Rizzuto and Pozzan (2006), this term (especially with regard to its spatial dimensions) has several diVerent meanings depending on one’s area of interest. In this chapter, however, like Rizzuto and Pozzan, we use the term in a general way to describe Ca2þ dynamics that do not involve the entire cell cytoplasm, but that remain localized to a specific cytoplasmic domain, or occur within a particular organelle or its periorganellar environment. Thus, one of the current challenges researchers are facing in the field of Ca2þ imaging is that of resolving changes in [Ca2þ] within, and between, various subcellular microdomains. An eVective strategy to address this challenge that is common to both fluorescenceand luminescence-based imaging techniques is to exclusively visualize Ca2þ dynamics in specific microdomains using genetically encoded and targeted Ca2þ reporters (GET-CRs). These come in two general forms, fluorescent GET-CRs and bioluminescent GET-CRs, respectively. At the other end of the size spectrum is the exciting prospect of imaging Ca2þ signals derived from GET-CRs within freely moving, large, organisms, for example, adult mice (Rogers et al., 2007). This presents a diVerent set of technical challenges to researchers in the Ca2þ imaging field. Fluorescent GET-CRs include the camgaroos (Baird et al., 1999; Griesbeck et al., 2001), G-CaMPs (Nakai et al., 2001; Ohkura et al., 2005), pericams (Nagai et al., 2001), case-sensors (Souslova et al., 2007), grafted EF-hands (Zou et al., 2007), and cameleon-types (Miyawaki et al., 1997; Ishii et al., 2006; Tsuruwaka et al., 2007; and reviewed by Zorov et al., 2004; McCombs and Palmer, 2008). Bioluminescent GET-CRs include single protein entities such as aequorin
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(Cheung et al., 2006; Torrecilla et al., 2000), obelin (Stepanyuk et al., 2005), mitrocomin (Inouye and Sahara, 2009), clytin (Inouye, 2008), and photina (Bovolenta et al., 2007). In addition, there is a growing number of aequorinderived bioluminescence resonance energy transfer (BRET)-based complexes such as the GFP-aequorins (Ashworth and Brennan, 2005; Baubet et al., 2000; Martin et al., 2007 ; Rogers et al., 2005, 2007), as well as other wavelength-shifted variants (Gorokhovatsky et al., 2004). To date, apoaequorin alone, or apoaequorin in tandem with another BRET protein, has been genetically expressed in a diverse range of diVerent species; either in the whole organism or in specific tissues within an intact organism (See Table I and Fig. 1). For example, apoaequorin has been ubiquitously expressed in whole zebrafish embryos (Cheung et al., 2006) or specifically targeted to the Malpighian tubules in Drosophila (Rosay et al., 1997), while the BRET complexes GFP-apoaequorin and YFPapoaequorin have been specifically targeted to neuronal cell subsets of Drosophila (Martin et al., 2007), and the endodermis and pericycle of Arabidopsis roots (Kiegle et al., 2000), respectively. Furthermore, apoaequorin or apoaequorinBRET complexes have been expressed either ubiquitously in the cytosol of cells in culture or, using specific targeting sequences, in distinct organelles of cells in culture (see Table II, and a recent review by Gerasimenko and Tepikin, 2005). Specific organelles targeted include: the ER (Montero et al., 1997), mitochondria (Rizzuto et al., 1992), the Golgi apparatus (Pinton et al., 1998), the nucleus (Brini et al., 1993, 1994), gap junctions (George et al., 1998), subplasma membrane domains (Marsault et al., 1997; Nakahashi et al., 1997), secretory vesicles (Mitchell et al., 2001), and the outer mantle of secretory granules (Pouli et al., 1998), to name but a few examples. In this chapter, we focus on describing the practical uses of bioluminescent GETCRs, especially those based on apoaequorin. In addition, we provide the reader (in table form) with a review of the literature to date listing representative examples of whole organisms, tissues, and cells that have been transfected with apoaequorin or an apoaequorin-BRET complex, as well as a list of organelles and subcellular domains that have been specifically targeted (see Tables I and II). We also summarize diVerent strategies used for loading various derivatives of the apoaequorin cofactor, coelenterazine (Shimomura et al., 1989) into cells, tissues, and intact organisms (summarized in Table III). Furthermore, we describe recent advances in detection and imaging technologies used to measure and visualize light generated by the aequorin-Ca2þ luminescent reaction within cells, tissues, and intact organisms (summarized in Table IV and illustrated in Figs. 2 and 3). Our hope is that this chapter will provide a starting point for researchers wishing to use GET-CRs to measure or visualize Ca2þ dynamics from cells, tissues, or intact organisms. Furthermore, the references provided in Tables I–IV should lead them to more detailed information regarding a biological system and/or experimental setup that will complement their own research interest. For loading holoaequorin into cells and embryos, we refer readers to the practical methodologies described in Miller et al. (1994), as
Table I Examples where apoaequorin or apoaequorin-BRET complexes have been targeted to a diverse range of diVerent species Kingdom Animalia
Plantae
Fungi
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Monera
Mus musculus (mouse)
Fish
Danio rerio (zebrafish)
Amphibians Insects
Xenopus laevis (African clawed frog) Drosophila melanogaster (Fruit fly)
Dicots
Nicotiana plumbaginifolia (Tobacco) Arabidopsis thaliana
Whole organism Mitochondrial matrixa Whole organismb Trunk musculature Plasma membrane of oocytes Malpighian tubules—diVerent cellular components Mushroom bodies and antennal lobesa Whole organism Whole organism
Solanum tuberosum (Potato) Triticum aestivum (Winter wheat) Physcomitrella patens Phyllosticta ampelicida Neurospora cassa Aspergillus awamori Aspergillus niger
Guard cells Endodermis and pericycle of rootsc Whole organism Whole organism Whole organism Whole organism Whole organism Whole organism Whole organism
Saccharomyces cerevisiae Schizosaccharomyces pombe Dictyostelium discoideum (Slime mold) Phaeodactylum tricornutum Escherichia coli Bacillus subtilis Streptococcus pneumoniae Anabaena strain sp. PCC7120
Whole organism Whole organism Whole organism Whole organism Whole organism Whole organism Whole organism Whole organism
Monocots Moss Funguses
Amoebozoa Diatoms Bacteria
Blue-green algae a
GFP-aequorin constructs were used. Transient transfection of apoaequorin mRNA was used. c YFP-aequorin constructs were used. b
Apoaequorin or apoaequorin-BRET targeted to:
Mammals
Yeast Protista
Species
References Yamano et al. (2007) Rogers et al. (2007) Cheung et al. (2006) Cheung (2009) Daguzan et al. (1995) Rosay et al. (1997) Martin et al. (2007) Knight et al. (1991a) Knight et al. (1995, 1996), Sedbrook et al. (1996) Dodd et al. (2006) Kiegle et al. (2000) Fisahn et al. (2004) Nagel-Volkmann et al. (2009) Russell et al. (1996) Shaw et al. (2001) Nelson et al. (2004) Nelson et al. (2004) Nelson et al. (2004), Bencˇina et al. (2005) Batiza et al. (1996) Deng et al. (2006) Cubitt et al. (1995) Falciatore et al. (2000) Knight et al. (1991b) Herbaud et al. (1998) Chapuy-Regaud et al. (2001) Torrecilla et al. (2000)
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Fig. 1 Examples of the spatial patterns of Ca2þ signals generated by whole organisms, or by specific tissues or subcellular organelles within intact organisms, where aequorin or GFP-aequorin were genetically expressed. (A) Newborn mice stably expressing GFP-aequorin targeted to the mitochondrial matrix were injected intraperitoneally with native coelenterazine and bioluminescence activity was recorded with the animals un-restrained and freely moving. These are consecutive video images on to which have been superimposed the corresponding bioluminescence images. Each panel represents 2 s of accumulated light. Scale bar is 5 mm. Reproduced with permission, from Rogers et al. (2007). (B) An 18somite stage (i.e., 18 h postfertilization (hpf)) zebrafish embryo that was injected with apoaequorin mRNA at the one-cell stage to transiently express apoaequorin throughout the whole embryo, and then
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not much has changed with respect to these particular techniques since Volume 40 of the Methods in Cell Biology series was published.
II. Expression of Apoaequorin, GFP-Apoaequorin, and Other Apoaequorin-Based Spectral Variants in Cells, Tissues, and Whole Organisms Microinjected holoaequorin has been used since the late 1960s for monitoring changes in [Ca2þ]i in diVerent cells and tissues. The earliest reports describe the use of holoaequorin to detect Ca2þ transients in muscle and nerve cells (Baker et al., 1971; Ridgway and Ashley, 1967) as well as during activation in medaka eggs (Ridgway et al., 1977). This approach is only practical, however, for introducing aequorin into giant cells and large embryos, which are easy to microinject. The more recent development, from the mid 1980s to early 1990s, of genetic engineering techniques to introduce and express apoaequorin (the protein moiety of aequorin) cDNA in cells, tissues, and whole organisms (Inouye et al., 1989; Knight et al., 1991a,b; Nakajima-Shimada et al., 1991; Prasher et al., 1985; Saran et al., 1994), as well as to target apoaequorin to specific organelles within cells (Brini et al., 1993; Rizzuto et al., 1992), has paved the way for aequorin to be used as the Ca2þ reporter of choice in many more biological systems today, from cells in culture to complex multicellular organisms. GFP-aequorin was developed approximately 10 years ago in order to improve the stability and light emission properties of aequorin for single-cell imaging (Baubet et al., 2000). Based on the naturally occurring phenomenon of BRET, GFP-aequorin emits a red-shifted light emission (l ¼ 509 nm) relative to that of aequorin alone (l ¼ 470 nm) in the presence of elevated free Ca2þ ion concentrations. GFP-aequorin has a number of advantages over aequorin for monitoring changes in cellular Ca2þ concentrations, including increased stability and total light output. Furthermore, the expression level and distribution of the GFP reflects the expression level and distribution of apoaequorin; thus, the expression of apoaequorin can be directly visualized in living cells or tissues. Although the incubated with f-coelenterazine starting at the 64-cell stage to reconstitute aequorin. Each panel represents 120 s of accumulated light and consecutive panels are stepped at 60-s intervals. Scale bar is 200 mm. (C) D. melanogaster (P[GAL4] OK107 line) stably expressing GFP-aequorin in the mushroom bodies. Exposed fly brains were incubated for >1 h at room temperature with native coelenterazine, prior to imaging. The first panel shows the whole brain and the localization of GFP in the mushroom bodies. The following panels show consecutive bioluminescent images, each panel representing 15 s of accumulated light, following treatment with 70 mM KCl to induce Kþ-depolarization. Scale bar is 100 mm. Reproduced with permission, from Martin et al. (2007). (D) Nine-day old seedlings of Arabidopsis thaliana (ecotype RLD1) that constitutively express apoaequorin were incubated in the dark overnight in coelenterazine solution. These images show the total Ca2þ-dependent bioluminescence recorded from seedlings exposed to air or to diVerent concentrations of ozone for 1 h. Scale bar is 5 mm. #John Wiley and Sons Ltd. Reproduced with permission, from Evans et al. (2005).
Table II Examples where apoaequorin or apoaequorin-BRET complexes have been targeted to the cytosol of cells in culture or to distinct intracellular organelles either in cells in culture or intact organisms Species (common name and/or scientific name)
Cytosolic expression of apoaequorin or apoaequorin-BRET
Cell type
Kingdom: Animalia Human (Homo ECV304 (umbilical sapiens) vein endothelial cells) Diploid fibroblasts
HEK-293 cells (embryonic kidney cell line)
Organelle(s) targeted
References
✓
Mitochondria
Transient expression
Lawrie et al. (1996)
(used indo 1-AM and fura 2-AM
Mitochondria
Padua et al. (1998)
✓
–
✓
–
1 cell cultures (Transient expression) Some transient and some stable expression Stable expression
✓
Nucleus
✓
ER lumen, mitochondrial matrix Nucleus, mitochondria, ER
HEK 293T cells
✓
HeLa (immortalized epithelial cell line)
✓ (used Fura-2)
Nucleus ER lumen
(used fura 2)
ER
✓
Mitochondria, Golgi, ER Golgi, ER Golgi
Normal adult and Hailey-Hailey disease keratinocytes Jurkat cells (immortalized T-lymphocytes)
Comments
(used indo-1) (used proton-induced X-ray emission) (used indo PE3 (AM))
ER
HSV gene transfer vector Transient expression
Expression of GA and RA via HSV gene transfer vector Stable expression Transient and stable expression 1 cell cultures (HSV gene transfer vector) Transient expression Stable expression 1 cell cultures (Transient expression) Transient expression
Sheu et al. (1993)
Button and Brownstein (1993) Chamero et al. (2002) Brini et al. (2005)
Manjarre´s et al. (2008)
Brini et al. (1993) Montero et al. (1995) Alonso et al. (1998)
Pinton et al. (2000) Missiaen et al. (2004) Behne et al. (2003)
Narayanan et al. (2003)
(continues)
Table II (continued ) Species (common name and/or scientific name)
Cytosolic expression of apoaequorin or apoaequorin-BRET
Cell type
Organelle(s) targeted
Comments
African green monkey (Simia aethiops)
Kidney COS cell cultures COS7 cells
✓
–
Transient expression
✓ ✓
Transient expression Transient expression
Cow (Bos taurus)
Bovine adrenal medulla chromaYn cells
(used fura 2)
ER Mitochondria þ outer mitochondrial membrane ER
(used fura 2)
Mitochondria
✓ (also Fura 4F)
ER, nucleus mitochondria,
✓
Nucleus
Secretory granules
✓
Mitochondrial matrix
✓
Mitochondria þ outer mitochondrial membrane
Mu¨ller glial cells of adult retinal explants
✓
–
A-11 (nonmetastatic) and 3LL (metastatic) Lewis lung cancer cell lines Soma and neurites from adult superior cervical ganglion neurons
✓
–
(used fura 2)
Mitochondria
Bovine adrenal zone glomerulosa cells
Mouse (Mus musculus)
1 cell cultures (HSV gene transfer vector) 1 cell cultures (HSV gene transfer vector) 1 cell cultures (HSV gene transfer vector) 1 cell cultures (HSV gene transfer vector) 1 cell cultures (AdV gene transfer vector) 1 cell cultures (Transient transfection) 1 cell cultures (Transient transfection) Explant culture. Expression of GA via AdV gene transfer vector Stable expression
1 cell cultures (HSV gene transfer vector)
References Button and Brownstein (1993) Kendall et al. (1994) Brandenburger et al. (1999) Alonso et al. (1998)
Montero et al. (2000)
Villalobos et al. (2002)
Chamero et al. (2002)
SantoDomingo et al. (2008) Brandenburger et al. (1996) Brandenburger et al. (1999) Agulhon et al. (2007)
Yoshida et al. (1998)
Nu´n˜ez et al. (2007)
Rat (Rattus norvegicus)
Pancreatic b-cells from intact islets
(used fura 2)
Mitochondria
Myoblasts isolated from the extensor digitorum longus muscle in mdx and normal C57B1/10 mice Myotubes isolated from hind leg muscles of mdx and C57BL101 mice C2C12 skeletal muscle cell line NIH 3T3 fibroblasts
✓
Sub-sarcolemma
✓
SR, mitochondria þ plasma membrane
1 cell cultures (Transient expression)
Robert et al. (2001a)
(used fura 2 AM)
Mitochondria
Stable expression
Challet et al. (2001)
(used Fura-2)
ER
Alonso et al. (1998)
✓
HSV gene transfer vector Transient expression Stable expression Transient expression and AdV gene transfer vector Explant culture (HSV gene transfer vector) Transient expression of GA 1 cell cultures (HSV gene transfer vector) 1 cell cultures (Transient expression) 1 cell cultures (Transient expression) 1 cell cultures (Transient expression)
Nakazaki et al. (1998) Mitchell et al. (2001)
MIN6 (pancreatic b-cell line)
(used fura 2 AM) (used fura 2 AM)
Outer surface of intracellular membranes Mitochondria ER, secretary vesicle
Intact pancreatic islets of Langerhans from Balb/c mice Neuro2A (neuroblastoma cells) Cerebellar granule cells
✓
Nuclear
✓
–
(used fura 2)
ER
Skeletal muscle myotubes
✓ (also used fura-2)
Mitochondria, nucleus, SR
SR and ER
✓
ER lumen and lumen of the terminal cisternae of the SR
Explant culture. Expression of GA via HSV gene transfer vector 1 cell cultures (Transient expression)
Quesada et al. (2008)
Basset et al. (2004)
Biagioli et al. (2005)
Villalobos et al. (2005)
Baubet et al. (2000) Alonso et al. (1998)
Brini et al. (1997)
Robert et al. (1998)
Brini et al. (2005)
(continues)
Table II (continued ) Species (common name and/or scientific name)
Cytosolic expression of apoaequorin or apoaequorin-BRET
Cell type
Organelle(s) targeted
L6 myogenic cell line Ventricular myocytes from neaonatal Wistar rats A7r5 cells (aortic smooth muscle cell line) Aortic smooth muscle cells
(used indo PE3 (AM)) ✓
ER Mitochondria
✓
Plasma membrane
(used fura 2 AM)
Mitochondria
Anterior pituitary cells
(used fura 2)
ER
GH3 cells (pituitary cell line)
(used fura 2)
ER
✓
Nucleus
✓
Nucleus, ER
(used fura 2)
ER
✓
Nucleus
✓
Secretory granule membrane Mitochondria Nucleus, mitochondria, ER
PC12 cells (Adrenal medulla pheachromocytoma cell line)
✓ ✓
INS-1 cells (derived from insulinsecreting pancreatic b-cell tumor)
✓
Mitochondria ER
Comments
References
Transient expression 1 cell cultures (Transient expression) Transient expression
Narayanan et al. (2003) Robert et al. (2001b)
1 cell cultures (Transient expression) 1 cell cultures (HSV gene transfer vector) HSV gene transfer vector HSV gene transfer vector Expression of GA via HSV gene transfer vector HSV gene transfer vector HSV gene transfer vector Transient expression
Szado et al. (2003)
Transient expression Expression of GA and RA via HSV gene transfer vector Stable expression Stable expression
Dı´az-Prieto et al. (2008) Manjarre´s et al. (2008)
Marsault et al. (1997)
Alonso et al. (1998)
Alonso et al. (1998) Chamero et al. (2002) Chamero et al. (2008)
Alonso et al. (1998) Chamero et al. (2002) Moreno et al. (2005)
Kennedy et al. (1996) Maechler et al. (1999)
✓
ER
Stable expression
Chan et al. (2004)
SR
Rembold et al. (1997)
CHO-K1 cells
✓
–
Explant culture (AdV gene transfer vector) Stable expression
CHO.T cells CHO cells
✓ ✓
Transient expression Transient expression
Schneider 2 (S2) cells
✓
Mitochondria Peroxisomes Mitochondria, ER –
Button and Brownstein (1993), Sanchez-Bueno et al. (1996) Rutter et al. (1996) Lasorsa et al. (2008)
Stable expression
Torfs et al. (2002)
Cells in suspension of [L]., cell line 6.6.12 Leaf discs
✓
–
Stable expression
Mitho¨fer et al. (1999)
✓
–
Stable expression
Cessna et al. (2000)
Cells in suspension
✓
–
Stable expression
Blume et al. (2000)
Whole organism
✓
Mitochondria
Stable expression
Greene et al. (2002)
Whole organism
✓
–
Stable expression
ER lumen Mitochondria
Stable expression Stable expression
Nakajima-Shimada et al. (1991) Strayle et al. (1999) Jung et al. (2004)
✓ ✓
Nucleus Mitochondria
Stable expression Stable expression
Xiong and Ruben (1996) Xiong et al. (1997)
H4-IIE cells (hepatoma cell line) Tail artery (from male Wistar rats) Hamster (Cricetulus griseus)
Fruit fly (Drosophila melanogaster) Kingdom: Plantae Soybean (Glycine max) Tobacco (Nicotiana tabacum) Parsley (Petroselinum crispum) Kingdom: Fungi Aspergillus nidulans Saccharomyces cerevisiae
Kingdom: Protista Trypanosoma brucei brucei
Procyclic cells
AdV, Adenovirus; HSV, Herpes Simplex Virus; CHO.T cells, CHO cells that over-express human insulin receptors; HEK 293T, 293 cells transformed with large T-antigen from SV40.
Table III Examples of the some of the reported coelenterazine loading protocols Species Kingdom: Animalia Mus musculis (Mouse)
Danio rerio (Zebrafish) Xenopus laevis (African clawed frog) Drosophila melanogaster (Fruit fly)
Kingdom: Plantae Nicotiana plumbaginifolia (Tobacco) Arabidopsis thaliana
Solanum tuberosum (Potato) Physcomitrella patens (Moss)
Kingdom: Fungi Neurospora crassa / Aspergillus niger / Aspergillus awamori Phyllosticta ampelicida Schizosaccharomyces pombe Saccharomyces cerevisiae
Coelenterazine loading protocol reported
Native coelenterazine was introduced into adult mice (at 4 mg/kg) by tail-vein injection and into new-born mice (at 2–4 mg/g) by intraperitoneal injection. Light emission was recorded immediately Minced tissues or cells of tissues were incubated with 0.2 ml RPMI 1640 containing 10 mM coelenterazine at 37 C for 5 h Embryos that had been dechorionated at the 64-cell stage were incubated with 50 mM f-coelenterazine, prepared in 30% Danieau’s solution Oocytes were incubated in 2.5 mM coelenterazine in a medium containing 5 mM b-mercaptoethanol Malpighian tubules from 4 to 14-day old adults were incubated in Schneider’s medium containing 2.5 mM coelenterazine for 4–6 h in the dark Exposed fly brains were incubated in fly ringers solution containing 5 mM native coelenterazine for >1 h at r.t. Seedlings were floated on water containing 2.5 mM coelenterazine o/n at r.t. in the dark Seedlings were submerged in 10 mM coelenterazine (which had been dissolved in ethanol) in dH2O for 7.5 h at r.t. in the dark Seedlings were floated on water containing 2.5 mM coelenterazine o/n at r.t. in the dark Plants were incubated in 5 mM hcp coelenterazine for 8 h Ground up moss tissue was incubated in 0.5 M NaCl, 5 mM mercaptoethanol, 5 mM EDTA, 0.1% gelatin, 10 mM Tris–HCl pH 7.4 containing 2.5 mM coelenterazine for 6 h or o/n Vogel’s medium containing 2.5 mM coelenterazine (prepared in methanol) was inoculated with 1 105 spores/ml. Inoculated medium was incubated for 24 h at 30 C in the dark 2.5 mM coelenterazine was pipetted over colonies growing in 1/2 potato dextrose agar and incubated for 4 h Cells were incubated in EMM medium containing 20 mM coelenterazine for 4 h at 30 C 0.1 volume of 590 mM coelenterazine (prepared in methanol) was added to 25–30 ml yeast culture and incubated for 20 min at r.t.
References Rogers et al. (2007)
Yamano et al. (2007) Cheung et al. (2006) Daguzan et al. (1995) Rosay et al. (1997) Martin et al. (2007)
Knight et al. (1991a, 1996) Sedbrook et al. (1996) Knight et al. (1996) Fisahn et al. (2004) Russell et al. (1996)
Nelson et al. (2004)
Shaw et al. (2001) Deng et al. (2006) Batiza et al. (1996)
Kingdom: Protista Dictyostelium discoidium (Slime mold) Kingdom: Monera Escherichia coli Bacillus subtilis Anabaena sp. Tissue Culture Cells H4-IIE cells Neuro2A cells HeLa cells Jurkat cells COS7 cells
Cells were incubated with coelenterazine solution to a final concentration of 50 mM for 24 h at r.t. in the dark
Cubitt et al. (1995)
Cells, diluted in 100 mM KCl, 1 mM MgCl2, Tris–HCl, pH 7.5, were incubated with coelenterazine (final concentration of 2.5 mM) o/n at r.t. in the dark Bacteria were incubated in TS medium containing 20 mg/ml kanamycin and 2.5 mM coelenterazine h for 1 h in the dark at r.t. Cells were incubated with coelenterazine (to a final concentration of 2.5 mM) for 4 h in the dark
Knight et al. (1991b)
Cells were incubated with 5 mM coelenterazine in Krebs–Ringer modified buVer containing 1 mM EGTA for 1 h at r.t. Cells were incubated with 5 mM coelenterazine, 10 mM b-mercaptoethanol and 4 mM EDTA in PBS for 2–4 h at 4 C Cells were incubated with 2.5 mM coelenterazine for 6–8 h at 37 C Cells were incubated with 5 mM coelenterazine-n for 2 h at 37 C Cells in suspension were incubated with 2.5 mM coelenterazine in DMEM o/n at 4 C
Chan et al. (2004)
r.t. is room temperature; o/n is overnight.
Herbaud et al. (1998) Torrecilla et al. (2000)
Baubet et al. (2000) Brini et al. (1993) Narayanan et al. (2003) Kendall et al. (1994)
Table IV Examples of the detectors used to image aequorin-generated luminescence Company
Type of detector reported
Berthold Technologies (U.K.) Ltd, Hertfordshire, UK Biospace Lab., Paris, France
LB980 intensified tube camera
Dittie Thermografie, Bonn, Germany
Giotto 1.12 microchannel plate-linked image intensifier tube (1st generation) Luminograph LB980 low-light camera system C2400-40H ICCD
EG&G, Berthold Technologies (U.K.) Ltd, Hertfordshire, UK Hamamatsu Photonics GmbH Deutschland, Herrsching, Germany Hamamatsu Photonics Co., Hamamatsu, Japan
Photek Ltd., East Sussex, UK
Cooled GaAs ICCD (3rd generation)
Ultrasensitive VIM camera system (a CCD camera equipped with an intensifier, Model C-1400-47) VIM photon counting camera
C2400-20M ICCD RA-IPD IPD 3
Photek 216 ICCD EDC-02 ICCD Photometrics, Tucson, AZ Photonic Science, Robertsbridge, UK
CH220 CCD imager Cooled extended ISIS video camera
Princeton Instruments, Trenton, NJ
TE/CCD512BKS CCD
Examples of specimens visualized
References
Nicotiana plumbaginifolia (tobacco) seedlings Mus musculus (mouse)—intact animals Triticum aestivum (winter wheat) seedlings Phyllosticta ampelicida
Wood et al. (2001)
Petroselinum crispum (parsley) suspension cultures Arabidopsis thaliana intact plants
Blume et al. (2000)
Bovine adrenal chromaYn cells Mouse superior cervical ganglion neurons Mouse intact pancreatic islets
Villalobos et al. (2002) Nu´n˜ez et al. (2007)
CHO.T cells H4-IIE cells Danio rerio (zebrafish) embryos Drosophila melanogaster (fruit fly) mushroom bodies Adult mouse retinal explants Arabidopsis thaliana seedlings Arabidopsis thaliana seedlings/ leaves Arabidopsis thaliana seedlings Neuro2A (mouse neuroblastoma cells) Nicotiana plumbaginifolia (tobacco) seedlings
Rogers et al. (2007) Nagel-Volkmann et al. (2009) Shaw et al. (2001)
Furuichi et al. (2001)
Villalobos et al. (2005), Quesada et al. (2008) Rutter et al. (1996) Chan et al. (2004) Cheung et al. (2006) Martin et al. (2007) Agulhon et al. (2007) Evans et al. (2005) Knight and Knight (2000), Grant et al. (2000) Sedbrook et al. (1996) Baubet et al. (2000) Sai and Johnson (2002)
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improved light emission properties of GFP-aequorin are still not completely understood, it is thought that they likely relate to an improved stability of the apoaequorin protein or to an increased quantum yield of the Ca2þ-activated photoprotein complex, or perhaps both. The cloned GFP-apoaequorin gene has been engineered to target diVerent subcellular compartments using similar strategies as those developed and described already for aequorin (Rizzuto et al., 1992). This approach has also been extended to include other spectral variants of these aequorin-based reporters, by replacement of the GFP gene with the sequences encoding Venus (YFP), mRFP, and more recently, mOrange (Bakayan et al., 2009; Curie et al., 2007; Manjarre´s et al., 2008). The development of these bifunctional reporters together with new imaging technologies (see Section IV) has considerably extended the number of applications possible with aequorin and in particular, has facilitated important advances in multicompartment measurements of Ca2þ concentrations and in noninvasive whole animal Ca2þ-imaging studies of the mammalian system.
A. Expression of Apoaequorin
1. Protocol 1: Preparation of Transgenic Zebrafish that Express Apoaequorin in a Tissue-Specific Manner (e.g., in the skeletal musculature) a. Materials piP-HE (apoaequorin plasmid; Inouye et al., 1989) ap-SK plasmid (a-actin promoter; Higashijima et al., 1997) pIRES2-EGFP plasmid (Clontech Laboratories, Inc., Mountain View, CA, USA) pCMVTNT vector (Promega Corp., Madison, WI, USA) f-coelenterazine (C-6779; Molecular Probes, Invitrogen Corp., Eugene, OR, USA) Methyl cellulose (M0387; Sigma–Aldrich Corp., MO, USA) b. Methods i. Preparation of the pa-KS-aeq-IRES-EGFP plasmid. To prepare the pa-KSaeq-IRES-EGFP plasmid, use PCR to amplify the apoaequorin gene from the piP-HE plasmid, with the following oligonucleotide primers: 50 -accagaattcatgacaagcaaacaatactcagtcaagcttacatcagac-30 and 50 -accagtcgacttaggggacagctccaccgtagag-30 , such that EcoR1 and Sal1, are added to the 50 and 30 ends of the apoaequorin gene, respectively. The apoaequorin gene can then be cloned into the pIRES2-EGFP plasmid using these restriction sites. Excise the aeq-IRES-EGFP fragment with EcoR1 and Not1, and then clone it into the ap-SK plasmid to obtain an aeqIRES-EGFP fragment with an a-actin promoter (i.e., a-aeq-IRES-EGFP). In parallel, amplify the SV40 late polyadenylation signal (pA) from the pCMVTNT vector using the following oligonucleotide primers: 50 -accagcggccgccagacatgataagatacattg30 and 50 -accagagctctctagaaccggttaccacatttgtagaggtttt-30 , adding Not1 to the 50 end,
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and Age1, Xba1, and Sac1 to the 30 end of the SV40 late polyadenylation signal. The SV40 late polyadenylation signal can then be cloned into the pBluescriptII-KSþ plasmid with Not1 and Sac1, after which the a-aeq-IRES-EGFP fragment can also be cloned into this plasmid using Xho1 and Not1. The enhanced green fluorescent protein (EGFP) marker gene is regulated by the IRES-sequence for the subsequent identification of transgenic fish. Use of the IRES-sequence enables the translation of both apoaequorin and the EGFP marker from a single mRNA; thus, the expression level and distribution of EGFP reflects the expression level and distribution of apoaequorin (Fahrenkrug et al., 1999; Wang et al., 2000). ii. Generation of transgenic zebrafish that express apoaequorin in the skeletal muscles. Linearize the pa-KS-aeq-IRES-EGFP plasmid with Xba1 and then microinject 1 nl (i.e., 100–200 pg) into the center of the blastodisc of the zebrafish embryos at the one-cell stage. The microinjection pipettes and pressure injection system used are described in detail in Webb et al. (1997). The injected embryos should then be maintained at 28.5 C and screened for the expression of EGFP after 24 hpf. Embryos (F0) that express EGFP should be raised to adulthood for further transgenic germ line screening. This involves: (1) The F0 fish being crossed with the wild-type fish to get the F1 generation. (2) If some of the F1 embryos express EGFP, then this indicates that one of their parents was transgenic (i.e., it was heterozygous). The F1 embryos that express EGFP can then be raised to adulthood and intercrossed with one another to produce the F2 generation. (3) In the F2 generation, 50% of the oVspring should be heterozygous, 25% should be homozygous, and 25% should be wild-type. (4) The homozygous F2 transgenic fish may then be identified by crossing the EGFP-expressing fish with wild-type fish; if all of the F3 oVspring express EGFP, then their transgenic parent was a homozygote; if 50% of the F3 embryos express EGFP, then their transgenic parent was heterozygote. The homozygous F2 fish can then be intercrossed with one another to obtain stable transgenic lines. iii. In vivo reconstitution of aequorin. Dechorionate the a-actin-apoaequorin transgenic embryos when they are at the eight-cell stage (we dechorionate embryos manually with watchmaker’s forceps) and incubate them in a custom-designed injection/imaging chamber (described in Webb et al., 1997) with 20 mg/ml f-coelenterazine in 30% Danieau’s solution to reconstitute the active aequorin. Prepare the f-coelenterazine as a stock solution of 2 mg/ml in methanol and dilute it in 30% Danieau’s solution just prior to use. In this transgenic F2 fish line, the EGFP and thus the apoaequorin, are expressed in the musculature at low levels at 12 hpf (i.e., the 6-somite stage) and the level of expression increases in an approximately linear manner up to 24 hpf. In addition, at 24 hpf EGFP and thus apoaequorin were expressed throughout the entire musculature, that is, in both the slow and fast muscles. Thus, this line of muscle-specific apoaequorin-expressing transgenic zebrafish can be used to visualize and characterize the Ca2þ signals generated in the trunk musculature during its formation and function. For imaging, these laterstage embryos may be immobilized with 3% methyl cellulose. We have collected
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both temporal and spatial information regarding the Ca2þ signals generated by the musculature from these embryos up to 96 hpf. For imaging, we use two custombuilt Photon Imaging Microscope Systems (PIMS; Science Wares, Falmouth, MA, USA), one based on an IPD-425 (Photek Ltd., Sussex, UK) and the other based on a back-illuminated EMCCD (DU-897 iXonEMþ camera) that was purchased from Andor Technology (Belfast, Northern Ireland, UK) and then optimized by Science Wares Inc. for detecting single photon events at the emission rates typical for aequorin-based imaging (see Section IV for further details).
B. Expression of GFP-Apoaequorin
2. Protocol 2: Transient and Stable Transfection of Mammalian Cells with GFP-Apoaequorin Using Plasmid DNA Many of the expression vectors designed for gene delivery are commercially available. They contain a number of units, including an immediate-early enhancer/ promoter sequence such as human cytomegalovirus (HCMV), a multiple cloning region for insertion of the reporter gene, an antibiotic resistance gene (e.g., Ampicillin) for selection of the vector in Escherichia coli, and an additional antibiotic resistance gene for selection in mammalian cells (e.g., Neomycin G148). For mammalian expression, a good starting point is to clone the reporter gene into a vector containing an HCMV promoter (Williams et al., 2005). The constitutive immediate-early HCMV promoter drives high levels of GFP-aequorin expression in many mammalian cell lines following transient transfection. Transfection reagents (e.g., cationic liposomes) enable recombinant DNA delivery into the nucleus of many immortalized cell types such as HEK293, HeLa, COS7, CHO and NIH/3T3 cells, with high eYciency. Following transient transfection, stable clones can then be isolated using a combination of drug selection (e.g., Neomycin (G418) resistance) and cell sorting using flow cytometry. On the other hand, primary cells (e.g., cortical neurons) are usually transfected with very low eYciency (i.e., less than 1–5%) using this method and better results can be obtained by using recombinant viral vectors for gene delivery (Rogers et al., 2005). a. Materials Transfection reagent (e.g., FugeneÒ6 reagent, Roche Applied Science; Lipofectamine, Invitrogen; PolyFect, Qiagen). Ultrapure plasmid DNA (1 mg/ml)—Plasmid DNA can be purified using a plasmid purification kit (e.g., QiagenÒpurification kits). Optimem media (Invitrogen) 35-mm Petri dishes or 8-chamber slides (e.g., those available from MatTek corp. or Ibidi Gmbh). Any culture dish will do providing the bottom of the dish is optimized for high-resolution microscopy on an inverted setup (e.g., dishes prepared with a glass coverslip mounted underneath a hole cut in the bottom).
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This allows the use of objective lenses with high numerical apertures for maximum light collection. Native or h-coelenterazine (supplied by Molecular Probes, Invitrogen, US or Interchim, France; 1-5 mM stock solution prepared in 100% ethanol).
b. Methods i. Preparation of cells that are transiently transfected with GFPapoaequorin. Healthy cell monolayers can be transfected when they are approximately 50–75% confluent and imaged within 24–48 h following transfection. Wash cells 1 and resuspend with serum-free medium (with no antibiotic/antimycotic) and then place cells back into the 37 C/5% CO2 incubator. For transfecting a 35mm dish, prepare a microcentrifuge tube containing 150 ml of serum-free media. Add 4.5 ml of transfection reagent (this amount may be increased according to the cell type or reagent used). Vortex the tube and then add 1.5 ml of plasmid DNA (1 mg/ml). Mix by flicking the tube and leave the tube at room temperature for approximately 30–45 min to allow the formation of a complex. Following incubation, remove the cells from the incubator and add 100 ml of the mix drop-wise and gently swirl the dish before placing back into the incubator. Although optional, healthy cells can be more easily maintained if the medium is changed after 6 h with fresh medium containing fetal calf serum (FCS). After 24–48 h, replace the normal growth medium with a serum-/phenol red-free medium (used for imaging), and incubate the cells for 1 h with 5 mM coelenterazine (native or h-according to the type of study). Single cells expressing high levels of GFP-aequorin can be selected by their GFP fluorescence, and used for bioluminescence Ca2þ imaging studies.
ii. Preparation of stable cell lines expressing GFP-apoaequorin. Follow the protocol for preparing cells for transient transfection (see previous section). At 48 h after transient transfection, start the selection process by adding a selective medium containing the appropriate antibiotic (e.g., Geneticin, or Puromycin). The antibiotic concentration used (starting at an upper concentration of 1 mg/ml) needs to be optimized for diVerent cell types. The medium should be changed with fresh selective medium every 2–3 days over a period of several weeks. During this time, the concentration of antibiotic may be gradually decreased. Isolation of GFP-positive clones into 96-well plates can then be facilitated using flow cytometry. In our experience, approximately 10% of the clones survive and proliferate after a first round of FACS sorting. Since stable transfection is a random integration event and a large amount of variability is expected, the clones should be selected based on the level of GFP intensity and homogeneity as well as cellular morphology, and where instrumentation is available (e.g., Nikon’s Biostation or the Incucyte from Essen Instruments), clones can also be selected based on growth curves.
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3. Protocol 3: Preparation and Transfection of Organotypic Brain Slices with Recombinant Adenovirus-5 Vector Containing the GFP-Apoaequorin Gene Recombinant viral vectors, such as human adenovirus serotype 5 (Ad5), adenoassociated virus (AAV), Sindbis viruses, or retroviruses (e.g., Lentiviruses), are highly eYcient expression vectors for gene delivery in mammalian cells or tissues (Tenenbaum et al., 2004). Both Ad5 and the Sindbis virus have been used to mediate high levels of expression of GFP-apoaequorin in primary neuronal cultures, brain slices, and retinal explant cultures (Rogers et al., 2005). Ad5 was found to preferentially infect glial cells in cortical- or hippocampal-derived tissue as well as Mu¨ller cells (a type of glial cell) in retinal explant cultures (Rogers et al., 2005).
a. Materials Vibratome (e.g., Model VT-1000, Leica) Gas tank (95% O2 / 5 % CO2) Glass petri dish (3–5 cm diameter) Membrane filter inserts (e.g., 12 mm TranswellÒ Permeable Supports, Corning) 12-well tissue culture plates Artificial cerebrospinal fluid (ACSF), pH 7.4 (124 mM NaCl, 3 mM KCl, 2 mM CaCl2, 1.3 mM MgCl2, 25 mM NaHCO3, 1.25 mM NaHPO4, and 10 mM glucose) Superglue, Blades, Low temperature melting Agar, Culture medium (50 % MEM, 25 % HBSS, 25 % Horse Serum, 6.5 mg/ml glucose, 2 mM L-glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin, pH 7.2). Ad5-GA Viral particles (15 x 108 particles). Specimen chamber for live imaging
b. Methods Organotypic slices can be prepared similarly to previously reported methods (Stoppini et al., 1991). Briefly, prepare 400-600 mm slices as described in section 3b. Once the slices are cut, gently transfer them using a Pasteur pipette onto a culture membrane insert and into a 12-well culture plate containing prewarmed culture medium. Slices can be kept in culture for 4–5 days and viral particles added directly to the medium approximately 48 h prior to imaging. After viral transfection and verification of GFP expression, the membrane culture insert with attached slice can be moved to a larger Petri dish containing growth media (e.g., 35 mm) and the membrane excised with a scalpel blade. Care should be taken to avoid folding of the membrane insert, which will cause injury to the tissue. The membrane with attached slice should then be carefully inserted into an imaging chamber and a tissue anchor placed on top to secure the tissue (e.g., Series RC-20, Harvard Apparatus). Once the slice is mounted onto an inverted microscope, a simple gravity flow system for delivering buVer together with a small peristaltic pump connected to the output can be used for perfusion (Mohammed et al., 2007).
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i. Transgenic mice expressing GFP-apoaequorin reporters. GFP-aequorin has also been expressed in a number of mammalian cell lines, as well as in organisms such as Plasmodium bergei (Billker et al., 2004), Drosophila melanogaster (Martin et al., 2007), and in mice (Rogers et al., 2007). Transgenic mice can provide a source of cells, tissues, or organs for studies ex vivo or for studies in vivo (Rogers et al., 2007). In the case of targeted reporters, these are especially useful because they provide information regarding the localization of any probe-derived signals, which would otherwise be diYcult because of the low to moderate resolution aVorded by bioluminescence imaging. In addition, an inducible or conditional expression system could be introduced into the vector (e.g., Cre/Lox or Tet inducible elements), to ensure the absence of a phenotype in the event that expressing the reporter ubiquitously from the early stages of development is found to be detrimental. A conditional expression system also enables expression to be activated in a specific cell population or at diVerent stages of development. Indeed, the UAS-Gal4 system enables the specific expression of GFP-aequorin in neuronal subsets of the fly brain, allowing specific neuroanatomical mapping of Ca2þ signaling pathways (Martin et al., 2007). Transgenic mice can be generated via one of two methods: (1) using a ‘classical’ transgenesis approach, where the transgene is randomly integrated into the genome (Constantini and Lacy, 1981), or (2) by homologous recombination, which enables directed integration of the transgene (e.g., knock-in of the HPRT locus; Rogers et al., 2007). Similarly to pronuclear injection, lentiviral vectors can be used to deliver the transgene into the fertilized mouse egg (Ikawa et al., 2003). However, these methods can result in random and multiple integrations of the transgene. In contrast, homologous recombination targets transgene insertion in a single-copy to a known site in the genome ensuring a more predictable expression pattern and phenotype based on the known integration site (Bronson et al., 1996). Transgenic mice conditionally expressing mitochondrially targeted GFP-apoaequorin have already been generated using this method (Rogers et al., 2007). Targeted insertion of the transgene was made 5’ to the X-linked hypoxanthine phosphoribosyltransferase (HPRT) locus (X-chromosome).
4. Protocol 4: Preparation of Acute Brain Slices from Transgenic Mice Expressing Mitochondrially Targeted GFP-Aequorin a. Materials Vibratome (e.g., Model VT-1000, Leica) Oxygen (95% O2/5% CO2) Glass petri dish (3–5 cm diameter) ACSF, pH 7.4 (124 mM NaCl, 3 mM KCl, 1.3 mM MgCl2, 25 mM NaHCO3, 1.25 mM NaHPO4 and 10 mM glucose) Superglue, Blades, Low temperature melting Agar Specimen chamber for live imaging (e.g., RC-20 chamber from Harvard apparatus)
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b. Methods ACSF should be prepared fresh on the day of experiments. Two 50 ml falcon tubes filled with ACSF (Ca2þ free) can be placed at 20 C for approximately 1–2 h prior to the preparation of brain slices. This partly frozen medium is used to fill the bath on the Vibratome where the brain will be sliced. Once the Vibratome is ready, rapidly remove the brains from neonates, dissect the cerebellum away, and place the brain ventrally against the agar block. Horizontal or coronal slices (400 mm) can be cut and transferred immediately to a small Petri dish containing oxygenated ACSF and coelenterazine (10 mM) and incubated at room temp, in the dark for 45–60 mins. Once the slice has been inserted into the imaging chamber, a slice anchor (Harvard Apparatus) can be used to secure the tissue. Slices should be continuously perfused (at a flow rate of 1 ml/min) with oxygenated ACSF containing 2 mM CaCl2. Bioluminescence signals can be monitored as previously described (Rogers et al., 2007). C. Summary of Section II The commercial development of products, kits, or services enabling genetic engineering of cells or animals means that these technologies are no longer out of reach to biologists who have experience in imaging but who have little molecular biology experience. BRET-based imaging depends on the degree of spectral overlap, relative orientation, and the distance between donor and acceptor dipoles. Fluorescent proteins derived from coelenterates or their variants are therefore likely to be the most suitable acceptors owing to their structural similarity with GFP (Tsien, 1998). The recent development of RFP-aequorin (Manjarre´s et al., 2008) has provided the means to simultaneously monitor Ca2þ signals from two diVerent microdomains within a single cell.
III. Introducing Coelenterazines into Cells, Tissues and Embryos Coelenterazine is the small ( 400 Da) prosthetic group that binds with apoaequorin to form the active aequorin complex. As coelenterazine is subject to oxidation, it is normally supplied in a sealed vial free of O2. Prior to reconstitution, the coelenterazine should be stored at 20 C. In addition, coelenterazine is poorly soluble in water and so stock solutions are normally prepared in methanol. In this form, coelenterazine is stable for 3 months at 20 C. Coelenterazine was originally isolated from Aequorea victoria; however, in the 1970s a procedure for chemically synthesizing coelenterazine was developed (Inoue et al., 1975; Kishi et al., 1972). This procedure has since been used for preparing coelenterazine and its analogs (Jones et al., 1999). Many of the coelenterazine analogs possess properties diVerent from those of native coelenterazine. These include half-life, aequorin regeneration rate, luminescence capacity, emission maximum and membrane permeability, the latter being due to the lipophilic nature of coelenterazine. For example, f-coelenterazine has the same half-life as native coelenterazine
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(Shimomura, 1991) but when it is reconstituted with apoaequorin to form an aequorin complex (f-aequorin) the level of luminescence produced on reaction with Ca2þ is almost 20-fold higher than that produced when native coelenterazine is used. In addition, f-coelenterazine has the highest permeability through cell membranes (Shimomura, 1997). As coelenterazine is lipophilic, apoaequorin-expressing cells, tissues, and whole organisms can simply be incubated in coelenterazine solution. However, this method is successful only in tissue culture cells and in simple organisms that have a large surface area-to-volume ratio where eYcient diVusion occurs. In more complex, multicellular organisms such as developing vertebrate embryos, reconstituting aequorin is more of a challenge. In the case of our a-actin-apoaequorin transgenic zebrafish, we started our f-coelenterazine incubation as early as the eight-cell stage (i.e., 1.25 hpf ) when the embryonic cells had a large surface area-to-volume ratio, and embryos were incubated continually in this 20 mg/ml coelenterazine both up until, and during data collection, which took place from 16 to 48 hpf. In the case of the apoaequorin-expressing transgenic mice, Rogers et al. (2007) introduced native coelenterazine into adult mice (at 4 mg/kg) by injection into the tail vein and into new-born mice (at 2–4 mg/g) by intraperitoneal injection. These, and other protocols used for introducing coelenterazine into various intact organisms and tissue culture cells are summarized in Table III.
IV. Techniques for Detecting Aequorin Luminescence Currently, several diVerent types of equipment are commercially available that can be used to detect or visualize aequorin-generated luminescence. These range in capability, price, design, and commercial availability. At the lower cost end, there is the simple test tube/culture dish luminometer, which provides only temporal Ca2þ signaling information, costs just a few hundred US dollars, and is supplied by several diVerent companies. At the higher cost end are several custom-designed imaging systems, which provide both temporal and spatial luminescent information, as well as bright-field and fluorescence images (if required), to enable the correlation of Ca2þ signaling events with morphological features and other cellular changes. These systems are obviously a lot more costly and are built to order by a small number of specialist companies. Some examples of the types of detectors that have been used to image aequorin-generated luminescence are shown in Table IV. It may be diYcult to justify the purchase of expensive single photon imaging equipment at early stages in a project. Often a photon counting photomultiplier tube (PMT) can be used in place of an imaging photon detector (IPD) to determine the timing and amplitude of bioluminescence signals in living systems. By adding a near-IR light source and an appropriate blocking filter in front of the PMT, a relatively inexpensive near-IR sensitive camera can be used to continuously monitor morphological development while the PMT reports total bioluminescence activity. An example of this type of system is shown in Fig. 2A. Accurate correlation
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of bioluminescence signals with morphological events facilitates planning, optimization, and standardization of protocols for subsequent imaging experiments. Over the past few years, several novel aequorin-based detection system designs have been reported. One is the two-channel luminometer, which was made for dual-wavelength aequorin measurements (see Fig. 2B). This system was designed by Manjarre´s et al., 2008 and built by Cairn Research (Faversham, UK). Here, the luminescence emission from two spectrally distinct aequorins (GFP-aequorin and RFP-aequorin) that are coexpressed in diVerent subcellular locations within the same cells is divided by a dichroic mirror and the resulting beams of light are filtered at 535 and 630 nm and then collected by two separate PMTs (Manjarre´s et al., 2008). Another new Ca2þ detection device is an imaging system capable of acquiring real-time bioluminescence data from living (and unrestrained) small animals such as mice (see Fig. 2C). This system was designed by Roncali et al. (2008). It is based around a Photon ImagerTM intensified CCD camera (Biospace Lab., France) operating in a photon counting mode. The ICCD camera is set on top of a lighttight chamber and records optical signals at a video rate of 25 Hz. Motion can also be monitored by using two cameras, one that records the signal of interest and the other that is used to video-track the animal. The latter can be achieved by illuminating the field of view with infrared light. The signals from both cameras are recorded simultaneously and electronically synchronized. A detailed description of this equipment is given by Roncali et al. (2008). A similar approach has been used with microscope-based imaging systems to continuously acquire bioluminescence image data emitted at short wavelengths while using longer wavelength illumination to simultaneously record transmitted light images that show morphology (Speksnijder et al., 1990). One of the most recent developments in bioluminescence detection involves using an EMCCD detector for single photon imaging (Martin et al., 2007; Rogers et al., 2008). The best bioluminescence imaging detectors are capable of single photon detection, and this requires that the detector somehow amplify the detected signal above background noise. All electronic imaging detectors ultimately convert incident photons from the sample into detected electrons,
information from cells expressing GFP- and RFP-aequorin in diVerent organelles. The system was designed by I. M. Manjarre´s, P. Chamero, M. T. Alonso, and J. Garcı´a-Sancho (Universidad de Valladolid and Consejo Superior de Investigaciones Cientı´ficas, Valladolid, Spain), and B. Domingo, F. Molina and J. Llopis (Universidad de Castilla-La Mancha, Albacete, Spain), and was built by Cairne Research (Faversham, UK). (C) A photon counting-based system, with a video monitoring function (via an IR-sensitive camera), for whole-body optical imaging of un-restrained, freely moving small animals, such as mice. The system was designed by E. Roncali, K. L. Rogers and B. Tavitian (Laboratiore d’Imagerie Mole´culaire Expe´rimentale, INSERM U803, Orsay, France) and M. Savinaud, O. Levrey and S. Maitrejean (Biospace Lab, Paris). Panels (B) and (C) are modified from Fig. 1 in Manjarre´s et al. (2008) and Roncali et al. (2008), respectively.
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which get amplified by an electronic circuit. The output of the circuit is monitored by a computer for recording purposes, and the collected data is then postprocessed to generate meaningful images (see Fig. 3). Bioluminescence can be diYcult to image because there is often very little signal, and because it is diYcult to predict exactly when and where the signal will be produced. Because the signals are often quite small, it is important to use the most eYcient optical system possible to collect the light. An overview of the main types of optical systems used for collecting light from bioluminescent samples is given by Karplus (2006) and see Fig. 4. In microscopy, it is important to select an objective with the highest ratio of numerical aperture to magnification (NA/mag). In macroimaging, it is important to select a lens with smallest possible working distance and the lowest possible f-stop, typically known as a ‘‘fast’’ lens. It can be helpful to have an electronic source on hand to test the eYciency of an optical system in bioluminescence imaging mode and ensure that the various components of the system are performing as expected (Cre´ton and JaVe, 2001). Another important consideration for bioluminescence imaging is that the sample being imaged must be kept in a light-tight enclosure to eliminate any direct or reflected ambient light from reaching the imaging detector, as it could easily overwhelm the bioluminescence signal. Because the sample is normally kept in complete darkness during bioluminescence imaging, it can be beneficial to periodically obtain bright-field and fluorescence images to determine the stage of development or morphological condition of the sample. Such bright-field and fluorescence images are typically orders of magnitude brighter than bioluminescent images, so they can be obtained in a very short period of time compared with that required to accumulate a meaningful bioluminescence image. Until the recent development of deep cooled, back-thinned, electron multiplying charge-coupled devices (EMCCDs), detectors capable of single photon imaging for bioluminescence were not well suited to acquiring bright-field and fluorescence images because they used microchannel plates to amplify and transmit images inside the detector. Microchannel plates blur and distort the
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images they transmit, have a limited dynamic range, and can also be permanently damaged if they are exposed to too much signal. While a nonintensified detector could be used to acquire bright-field and fluorescence images, the problem of blur and distortion in the bioluminescence image would still remain. In addition, it was a challenge to adjust the size and registration of images acquired from two diVerent detectors. An EMCCD sensor can be fabricated on a substrate alongside one or more amplifiers with programmable gain settings, so a computer can rapidly change from a low-gain to a high-gain setting. Thus, a computer-controlled EMCCD is capable of acquiring bright-field and fluorescence images at low-gain settings, as well as bioluminescence images at high-gain settings (see Fig. 4). This makes it possible to eliminate blurring and distortion in all three types of images to be acquired, improving the spatial resolution in the bioluminescence images by 2–3 times over what can be achieved with a microchannel plate based detector. Because all three types of images can be obtained with the same sensor, the scale and registration are identical as well. Furthermore, back-thinned EMCCDs have a significantly higher quantum eYciency in the visible light spectrum, compared with photocathode materials used in intensifier-based detectors, so they are able to respond more eYciently to weak signals. While EMCCDs are capable of detecting single photon events when the electron multiplying gain is high enough to overcome the read noise of the output amplifier, the electron multiplying gain mechanism is subject to substantial statistical variation. For example, when an output signal of 1000 electrons occurs, it is easily detected as a meaningful event, but it is not possible to be certain how many input electrons generated this signal—it may have been just 1, or 2, or 5 input electrons. As a result, EMCCDs operating in photon counting mode have limited ability to track large intensity changes. The maximum signal intensity that can be recorded reliably is essentially determined by the frame rate at which the sensor is read out. The range can be extended at the expense of the field of view by selecting a small region of interest, and/or at the expense of spatial resolution by binning together adjacent pixels on the sensor. Two additional limitations of EMCCDs arise from the circuitry used to read out the image data. First, in order to record the signal detected by the CCD sensor,
systems, respectively. The EMCCD can be modified to acquire bioluminescence information as well as bright-field and fluorescence images. On the other hand, a resistive-anode Imaging Photon Detector (RA-IPD) can be used in conjunction with a CCD camera, the latter to acquire bright-field and fluorescent images, when a higher dynamic range and temporal resolution are needed for the bioluminescence signal. For both the EMCCD and RA-IPD-based imaging systems, a high level of automation for the microscope makes it possible to rapidly switch between the various imaging modes, and also makes it possible to have the computer run automated acquisition sequences over extended periods, typically overnight. The motorized focus allows the computer to acquire image stacks in any imaging mode for three dimensional reconstructions. Both systems were designed and built by Science Wares Inc., Falmouth, MA, USA.
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there must be a period of dead time while the accumulated image data is shifted out of the active area of the sensor. Second, the signals used to shift the image data from the active area into the readout amplifier can also impose noise on the output that looks identical to single photon events in the photon counting mode. Significant advances have been made to minimize the noise (called clock-induced charge) that is generated. However, these two eVects are still significant such that increasing the readout rate beyond a certain level actually degrades the net single photon imaging performance of the EMCCD. In situations where significant bioluminescence intensity changes are taking place over periods less than a second, an intensifier-based detector is likely to be a better choice than an EMCCD (see Fig. 4). The spatial resolution of commercially available intensifier-based detectors is usually in the order of tens of microns at the detector input window, which is adequate for many applications, but not as good as what can be achieved with commercially available EMCCD detectors, which typically have a pixel size of 8 or 16 mm. There are two main types of intensifier-based detectors, those with a phosphor image output that is optically coupled to a visible light CCD, and those with an electrically encoded anode that produces position sensitive pulses for each detected event. The temporal resolution of detectors with an optically coupled phosphor output is typically in the range of tens of milliseconds due to the persistence time of the phosphor and the frame rate of the CCD. The best temporal resolution for single photon imaging can be in the order of tens of nanoseconds, and is achieved only by intensifier-based detectors with an electrically encoded anode. The dynamic range of such detectors is constrained by the dead time of the pulse processing electronics, which is typically in the order of a few microseconds per detected event. Improvements continue to be made in the spatial resolution of microchannel plates and throughput speed of encoded anode detectors (Lapington, 2004; Siegmund et al., 2005), but the cost and complexity of operating such detectors has prevented them from being used widely for bioluminescence imaging so far. Another factor that should be considered when selecting a detector for bioluminescence imaging is the expected signal-to-noise ratio of the recorded image data (Karplus, 2006). Frequently, accepting lower spatial resolution can result in a better signal-to-noise ratio. In situations where a fast or brief signal needs to be identified with high temporal accuracy, a photocathode detector is often capable of a better signal-to-noise ratio than an EMCCD. Even though an EMCCD detector can have 2–20 higher quantum eYciency than a detector with a photocathode, at the high readout rates needed for good temporal resolution, incident photons can still be lost during the dead time needed to transfer image data into the readout frame of the EMCCD, and the photons that are detected can be obscured by clockinduced readout noise. Figure 5 shows a comparison of the bioluminescence images acquired by an EMCCD and an RA-IPD photon imaging system at two diVerent stages of zebrafish development.
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V. Conclusions Living cells, tissues and whole organisms are essentially defined by their complex spatial structures. Underlying such broad morphological characteristics are much finer molecular assemblies, such as microdomains within the cytoskeleton, plasma membrane, nucleus and cytoplasm. It is becoming clear that such microdomains are the loci of many key signaling events, including those that involve Ca2þ and as such, they are becoming a major area of interest for investigators in the Ca2þ signaling field. In recent years, researchers have made spectacular advances on the live-cell imaging front, due in part to the development of techniques that combine breaking Ernst Abbe’s diVraction barrier (Abbe, 1873) being compatible with examining living systems. Such techniques include stimulated emission depletion (STED) microscopy (Hell, 2007), photoactivated localization microscopy (PALM) (Betzig et al., 2006), and stochastic optical reconstruction microscopy (STORM) (Rust et al., 2006). It is imperative, therefore, that Ca2þ imaging also joins the super-resolution revolution, and indeed significant progress has been made on this front with the development of fluorescence-based techniques such as single channel Ca2þ nanoscale resolution (SCCaNR) microscopy (Wiltgen et al., 2009). The continued development of both fluorescent and luminescent GET-CRs will undoubtedly also make a contribution to this advancement, especially if, in the case of the latter, the intensity of the Ca2þmediated luminescent emission can be increased. We find ourselves, therefore, at a most exciting and opportune time to extend our understanding of Ca2þ signaling in living cells from the microscopic to the nanoscopic level.
Acknowledgments We thank Philippe Bruˆlet, Marc Knight, and Jean-Rene´ Martin, who kindly gave us permission to use their previously published work. Special thanks also to Osamu Shimomura for his generous support of aequorin-based imaging over the years. We acknowledge financial support from Hong Kong RGC GRF grants: HKUST-6241/04M,-6416/06M,-661707 and-662109.
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INDEX
1,2-Bis(o-aminophenoxy)ethane-N,N,N’, N’-tetraacetic acid (BAPTA), 17–18 ionic strength dependent, 5, 7 pH dependent, 4 structural formulas for, 5 temperature, apparent aYnity, 6, 7 2P excitation microscopy Fluo-based dyes, 248, 249 Rhod-dyes, 249, 250
A Acetoxymethyl (AM) ester, 120 Aequoria victoria, 154 Aequorins apoaequorin expression, 268, 277–279 bioluminescent GETCRs, 265 BRET protein complex, 265 coelenterazine, 283–284 expression of GFP-Apoaequorin, 279–283 luminescence bioluminescence imaging, 287 ca 2þdetection device, 286 detectors types, 267 EMCCD detector, 286, 288 equipment, 284 factors, 290 intensifier-based detector, 290 photon counting photomultiplier, 284 schematic representations, 285 single photon imaging, 290 two-channel luminometer, 286 mitochondrial target, 160 strategies, 265 AM. See Acetoxymethyl (AM) ester Apoaequorin BRET complexes cytosol cell culture, 269–273 diverse range species, 266 materials, 277 methods in vivo reconstitution, 278
p -KS-aeq-IRES-EGFP plasmid preparation, 277–278 transgenic zebrafish generation, 278–279 Azid-1, 33–35
B Bioluminescence EMCCD detector, 286 fluorescent GET-CRs, 264 GETCRs, 265 optical systems, 287 single photon detection, 286 vs. EMCCD and an RA-IPD photon imaging system, 291 Bioluminescence resonance energy transfer (BRET), 255 aequorins, 265 cytosol cell culture, 269–273 diverse range species, 266 Blue fluorescent protein (BFP), 155 BRET. See Bioluminescence resonance energy transfer
C Ca2þ binding compounds, 104, 105 Ca2þ buVers association constant (KCa), 7, 10 BAPTA family, 5–7 basic steps, solution preparation, 16–18 dissociation constant (Kd), 3 EGTA, 4–6 fluorescent indicators, 3–4 ionic strength corrections, 11–12 measurement, free [Ca2þ], 12–13 Michaelis-Menten form, 9 potential complications, 18–19 proton activity coeYcient, 12 software programs, 19–23 spreadsheet for calibration calculations, 13–16 stability constants, 3 temperature corrections, 10–11 Ca2þ flux measurements, 99, 100, 108
301
302
Index Ca2þ in mitochondria, measurement cytosolic and mitochondrial Ca2þ, 146 Rhod-2 indicator, 143–145 Rhod-2, cytosol, 145–146 Ca2þ, manipulation buVering changes, 130–132 divalent Cation Ionophores, 129–130 extracellular buVers BAPTA, 126–127 EGTA, 126–127 lowering Extracellular [Ca2þ], 127–129 Ca2þ-selective electrodes dissociation constant, measurements of, 70 dynamic range of, 69 ETH 129, 86 key advantage, 68 microelectrodes (MEs) bath calibration, 81 Ca2þ-selective ligand, preparation and use of, 78–80 calibration procedure, 81–82 double-barreled, 80–81 electrolyte filling of, 78 ETH 1001, ionophore, 82 extracellular [Ca2þ], measurement, 83–85 glass tubing preparation, 76 microelectrode pulling and silanization, 77 solution perfusion, 81 troubleshooting, 85–86 minielectrode application of, 75–76 electrode potential of, 73 inhomogeneities, eVect of, 75 lifetime of, 72 resistance of, 72 response times of, 73–74 selective ligand, preparation and use, 71–72 storage of, 75 Nicolski-Eisenman equation, 68 Ca2þ-selective liquid membranes, coeVcients, 96, 97 Ca2þselective microelectrodes (CaSMs) construction microelectrode, 94 micropipette fabrication, 93 silanization, 93 properties response time, 97–98 response to Ion Activity, 94–96 selectivity, 96 spatial resolution, 96–97 self referencing calculation of flux, 103 correction for Ca2þ buVering, 104
diVerential concentration determination, 101–102 diVerential concentration measurement, 98–101 measurement of voltage gradients, 104–105 positional artifacts, 105–107 Caged Ca2þ chelators. See Photolabile Ca2þ chelators Calcium release-activated channels (CRAC), 185, 194–195 Cameleon, genetically encoded calcium sensors CFP/citrine couple, 159 ECFP/EYFP-based cameleons, 161 F46L mutation, 160 FIP-CBsm, 156 FRET, 156 mechanism, 161–163 myosin light chain kinase, 155 YC concentrations, 159 YC2.12 fluorescence, 160 YCX.60, 161 CaSM. See Ca2þselective microelectrodes CaSMs, construction microelectrode construction, 94 micropipette fabrication, 93 silanization, 93 CaSMs, properties Response Time, 97–98 Response to Ion Activity, 94–96 Selectivity, 96 Spatial Resolution, 96–97 CaSMs, self referencing calculation of flux, 103 correction for Ca2þ buVering, 104 diVerential concentration determination, 101–102 diVerential concentration measurement, 98–101 measurement of voltage gradients, 104–105 positional artifacts, 105–107 Cell-attached patch recordings, 192–193 Coelenterazine, 283–284 Confocal and multiphoton imaging absorbance/quantum yield, 246–247 advantages and disadvantages, 241–242 calibration of single wavelength dyes, 251–252 conversion of increments, 251 fluorescence lifetime, 247 Fmax values estimation, 252–253 Fmin estimation, 253 Fo¨rster resonance energy transfer microscopy acceptor photobleaching, 236 cameleon, 235, 237 donor quenching, 236 eYciency, 246, 247
303
Index FRET-FLIM approach, 236 GFP, 235–236 pericams, 237 intrinsic and dye fluorescence, 253–255 laser scanning confocal microscopy 2D frame scan, 231 Jablonski diagram, 232, 233 signal-to-noise, 231 Stokes shift, 232 limitations in speed, 230 multimodal and multiple fluorophore Di-8-ANEPPS, 256, 257 excitation-contraction coupling, 255 Fura dyes, 258 membrane voltage, 256 overlapping excitation spectra, 256 RH-237 emission spectrum, 257 multiphoton excitation laser scanning microscopy 2P excitation microscopy, 242, 243 biophysical perspectives, 242 high repetition rates, 245 IR light, 244 NA objectives, 228 parallel scanning confocal systems, 238 programmable matrix microscopy digital micromirror device, 239 filtering patterns, 239 liquid crystal display, 240 PSF, 229 signal detection system, 227, 228 spatial resolution, 229 spectral shift, 247 spinning disk confocal microscopy, 238–239 total internal reflection fluorescence microscopy evanescent wave, 234 refractive index, 233, 234 use of dyes 2P excitation microscopy, 248–250 single-photon confocal microscopy, 248 CRAC. See Calcium release-activated channels Cytoplasmic [Ca2þ]i, regulation of, 57 D Diazo compounds, Ca2þ chelators chemical properties, 39–41 photolysis, eVects of, 41–42 Dibromo-BAPTA (Br2-BAPTA), 4, 11 pH dependent, apparent aYnities, 4 structural formulas for, 5 temperature and ionic strength, apparent Ca2þ aYnity, 5
Digital micromirror device (DMD), 239 DM-nitrophen, Ca2þ chelators [Ca2þ]i changes, 37–39 absorbance of, 36 Ca2þ-and Mg2þ-aYnities of, 36 caged Mg2þ chelator, 36 kinetic behavior of, 38 quantum eYciency, 35 structure of and reaction scheme, 35 Dual-wavelength ratiometric dyes, 114, 115, 118–120
E Electrode calibration curves, 15, 16 Electron multiplying charge-coupled devices (EMCCD) bioluminescence detection, 286 computer-controlled EMCCD, 289 limitations, 289 single photon events, 289 vs. RA-IPD photon imaging system, 290, 291 EMCCD. See Electron multiplying chargecoupled devices Enhanced yellow fluorescent protein (EYFP) circular permutation, 163–164 Ethylene glycol bis( -aminoethylether)-N,N,N’, N’-tetraacetic acid (EGTA), 17–18 ionic strength, apparent aYnity, 6 pH dependent, 4–5 structural formulas for, 5 temperature, apparent aYnity, 6 Excel spreadsheet, Ca2þ calibration buVers, 14, 15 Excitation spectra Fluo-3, 117 Fura-2, 117
F Fluorescence lifetime imaging (FLIM), 236 Fluorescent Ca2þ Indicators dual-wavelength ratiometric dyes, 114, 115, 118–120 single-wavelength nonratiometric dyes, 114, 115, 118–120 Fluorescent Ca2þ Indicators, properties, 115 Fo¨rster resonance energy transfer (FRET) microscopy acceptor photobleaching, 236 advantage and disadvantage, 236 cameleon, 235, 237 donor quenching, 236 eYciency, 246, 247
304
Index Fo¨rster resonance energy transfer (FRET) microscopy (cont.) FRET-FLIM approach, 236 GFP, 235–236 pericams, 237 Fura-2, 33
G Genetically encoded calcium sensors Aequoria victoria, 154 Cameleon family CFP/citrine couple, 159 ECFP/EYFP-based cameleons, 161 F46L mutation, 160 origin, 155, 157 sensor mechanism, 161–163 YC concentrations, 159 YC2.12 fluorescence, 160 YCX.60, 161 yellow fluorescent protein, 159 Camgaroos, 163–164 cases 12 and 16, 167 GCaMPs, 165–167 green fluorescent protein, 154 pericam, 164–165 subcellular locations endoplasmic reticulum, 168 golgi, 169 mitochondria, 168 peroxisome, 169 plasma membrane, 169 tissue-specific expression comparative studies, 174–176 GCaMP, 172–173 inverse pericam, 171–172 TN-L15, TN-XL, 173, 174 TN-XXL, 174 YC2.1, 169–170 YC3.3er (citrine-based sensor), 171 uses, 176–177 GFP-apoaequorin recombinant viral vectors materials, 281 methods, 281–282 transgenic mice expressing mitochondrially materials, 282 methods, 283 transient and stable transfection materials, 279–280 methods, 280 Green fluorescent protein, 154
H Human cytomregalovirus (HCMV), 279
I Icrac. See Calcium release-activated currents Imaging photon detector (IPD), 284 Indicator fluorescence signal, conversion nonratiometric fluorescent indicator, calibration, 133–134 ratiometric fluorescent indicator, calibration, 134–138 Indicators, loading in the cells, 121–122 aqueous Solubility of AM Esters, 121 dye compartmentalization assessing extent of compartmentalization, 122–124 minimizing compartmentalization, 121–122 dye leakage, 124–125 procedure, 124–125 Inositol 1,4,5-trisphosphate receptors (IP3R) ATP ligands, 198 behavior analysis, 203 Ca 2þ signals, 190 Ca2þ ligands, 198 cell analysis, 195–196 current–voltage relationship, 204 cytopasm-out configuration, 202 DT40 cell expression, 193 electrical recording, 193 endoplasmic reticulum, 190 inner nuclear membrane expression, 198 intrinsic pore open, 190 IP3, 198 nuclear path-clamp recording, 194 single-channel recording, 191, 193 whole-cell recordings, 192 Intracellular Ca2þ confocal and multiphoton imaging 2P excitation microscopy, 248–250 advantages and disadvantages, 241–242 Fmax values estimation, 252–253 Fmin estimation, 253 FRET, 235–237 indicators, 245–247 intrinsic and dye fluorescence, 253–255 limitations in speed, 230 LSCM, 230–233 multimodal and multiple fluorophore, 255–259
305
Index multiphoton excitation laser scanning microscopy, 242–245 parallel scanning confocal systems, 238 programmable matrix microscopy, 239–241 single wavelength dyes, 251–252 single-photon confocal microscopy, 248 spinning disk confocal microscopy, 238–239 TIRF, 233–234 patch clamp methods, 185 Intracellular calcium signals fluorescent Ca2þ Indicators Ion channel modulation, Ca2þ chelators, 49–52 calcium channels, 51–52 potassium and nonspecific cation channels, 49–51
J Jablonski diagram, 232, 233
L Laser scanning confocal microscopy (LSCM) 2D frame scan, 231 Jablonski diagram, 232, 233 signal-to-noise, 231 Stokes shift, 232 Liquid crystal display (LCD), 240
M MaxChelator, 22 Microelectrodes (MEs), Ca2þ-selective bath calibration, 81 Ca2þ-selective ligand, preparation and use of, 78–80 calibration procedure, 81–82 double-barreled, 80–81 electrolyte filling of, 78 ETH 1001, ionophore, 82 extracellular [Ca2þ], measurement, 83–85 glass tubing preparation, 76 microelectrode pulling and silanization, 77 solution perfusion, 81 troubleshooting, 85–86 Minielectrode, Ca2þ-selective application of, 75–76 electrode potential of, 73 inhomogeneities, eVect of, 75 lifetime of, 72 resistance of, 72 response times of, 73–74
selective ligand, preparation and use, 71–72 storage of, 75 Multiphoton excitation laser scanning microscopy 2P excitation microscopy, 242, 243 biophysical perspectives, 242 high repetition rates, 245 IR light, 244 Muscle contraction, Ca2þ chelators, 52–53 Myosin light chain kinase (MLCK), 155
N Nipkow spinning disk, 239, 240 Nitr compounds, Ca2þ chelators [Ca2þ]i changes, cells, 33–34 azid-1, 33 BAPTA, 29, 30 fura-2, 33 nitr-5, 30–31 nitr-7, 30–31 nitr-8, 30–31 Nitr-5, 30–31 Nitr-7, 30–31 Nitr-8, 30–31 Nuclear patch-clamp recording conventional techniques, 193 ER membrane, 195 IP3R, 195–196 methods asymmetric recording solutions, 201 cytoplasm-out configuration, 202 DT40 Cell culture, 196–197 equipments, 199 nuclei isolation, 197–198 optimal filtering frequency, 201 pipette tip approaches, 201 recording configuration, 201 single channel record analysis, 202–208 solutions, 198–199
P Patch clamp methods calcium-selective channels, 184 intracellular calcium, 185 materials, 195 methods calcium release-activated currents, 194–195 cell-attached patch recordings, 192–193 fire-polishing pipettes, 191 giga-ohm seals, 190–192
306
Index Patch clamp methods (cont.) perforated patch recordings, 193–194 rig assemble, 187–188 Sylgard application, 188, 189 principles, 186–187 recombinant channels, 185, 186 Perforated patch recordings, 193–194 Photolabile Ca2þ chelators, 33 biological applications cytoplasmic [Ca2þ]i, regulation of, 57 filopodial activity, control of, 58 ion channel modulation, 49–52 muscle contraction, 52–53 rate-limiting steps, 58 synaptic function, 53–57 calibration, 45–48 diazo compounds chemical properties, 39–41 photolysis, eVects of, 41–42 DM-nitrophen [Ca2þ]i changes, 37–39 absorbance of, 36 Ca2þ-and Mg2þ-aYnities of, 36 caged Mg2þ chelator, 36 kinetic behavior of, 38 quantum eYciency, 35 structure of and reaction scheme, 35 introduction into cells, 42–43 light sources, 43–45 nitr compounds [Ca2þ]i changes, cells, 33–34 azid-1, 33 BAPTA, 29, 30 fura-2, 33 nitr-5, 30–31 nitr-7, 30–31 nitr-8, 30–31 properties of, 32 purity and toxicity, 48–49 Photomultiplier tube (PMT), 227, 231 Photon counting photomultiplier (PMT), 284 PMT. See Photon counting photomultiplier Point spread function (PSF), 229 Programmable matrix microscopy digital micromirror device, 239 filtering patterns, 239 liquid crystal display, 240 Proton activity coeYcient, 12
principle, 114, 115, 118–120 Recombinant viral vectors materials, 281 methods, 281–282
S Scatchard plot analysis, 13, 15, 16 Shuttle buVers, 104, 105 Single channel recording current-amplitude histograms, 203 dwell-time histogram, 207 electrophysiological records, 202 IP3R cation-selectivity, 206 kinetic analyses, 206 maximum interval likelihood method, 207 Mg 2þ, 198 mutations, 205 open probability, 208 Sigworth–Sine transformation, 207 stability plot, 206 Single-photon confocal microscopy, 248 Single-wavelength nonratiometric dyes, 114, 115, 118–120 SOCE. See Store-operated calcium entry Software programs, Ca2þ buVers, 19–20 accuracy, 21 ideal software criteria, 20 javascript web versions, 22–23 MaxChelator, 22 use and adaptability, ease of, 21 Store-operated calcium entry (SOCE), 185 Synaptic function, Ca2þ chelators, 53–57
T Tissue-specific expression comparative studies, 174–176 GCaMP, 172–173 inverse pericam, 171–172 TN-L15, TN-XL, 173, 174 TN-XXL, 174 YC2.1, 169–170 YC3.3er (citrine-based sensor), 171 Total internal reflection fluorescence (TIRF) microscopy evanescent wave, 234 refractive index, 233, 234
R Ratiometric fluorescent indicators Fura-2, 117 Indo-1, 117
Y Yellow fluorescent protein, 159
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