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Calcium Entry Channels
in Non-Excitable Cells
 METHODS IN SIGNAL TRANSDUCTION
SERIES

Joseph Eichberg, Jr. and Michael X. Zhu

Series Editors

Published Titles

Calcium Entry Channels in Non-Excitable Cells, Juliusz Ashot Kozak and James W.
Putney, Jr.
Lipid-Mediated Signaling Transduction, Second Edition, Eric Murphy, Thad
Rosenberger, and Mikhail Golovko
Signaling Mechanisms Regulating T Cell Diversity and Function, Jonathan Soboloff
and Dietmar J. Kappes
Gap Junction Channels and Hemichannels, Donglin Bai and Juan C. Sáez
Cyclic Nucleotide Signaling, Xiaodong Cheng
TRP Channels, Michael Xi Zhu
Lipid-Mediated Signaling, Eric J. Murphy and Thad A. Rosenberger
Signaling by Toll-Like Receptors, Gregory W. Konat
Signal Transduction in the Retina, Steven J. Fliesler and Oleg G. Kisselev
Analysis of Growth Factor Signaling in Embryos, Malcolm Whitman and Amy K. Sater
Calcium Signaling, Second Edition, James W. Putney, Jr.
G Protein-Coupled Receptors: Structure, Function, and Ligand Screening,
Tatsuya Haga and Shigeki Takeda
G Protein-Coupled Receptors, Tatsuya Haga and Gabriel Berstein
Signaling Through Cell Adhesion Molecules, Jun-Lin Guan
G Proteins: Techniques of Analysis, David R. Manning
Lipid Second Messengers, Suzanne G. Laychock and Ronald P. Rubin
Calcium Entry Channels
in Non-Excitable Cells

Edited by
Juliusz Ashot Kozak and James W. Putney, Jr.
CRC Press
Taylor & Francis Group
6000 Broken Sound Parkway NW, Suite 300
Boca Raton, FL 33487-2742

©2017 by Taylor & Francis Group, LLC

CRC Press is an imprint of Taylor & Francis Group, an Informa business

No claim to original U.S. Government works

Printed on acid-free paper

International Standard Book Number-13: 978-1-4987-5272-5 (Hardback)

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Library of Congress Cataloging-in-Publication Data

Names: Kozak, Juliusz Ashot, editor. | Putney, James W., Jr., editor.
Title: Calcium entry channels in non-excitable cells / [edited by] Juliusz
Ashot Kozak and James W. Putney, Jr.
Other titles: Methods in signal transduction.
Description: Boca Raton : Taylor & Francis, 2017. | Series: Methods in signal
transduction series | Includes bibliographical references and index.
Identifiers: LCCN 2016053681 | ISBN 9781498752725 (hardback : alk. paper)
Subjects: | MESH: Calcium Channels--physiology | Calcium Signaling--physiology
Classification: LCC QP552.C24 | NLM QU 55.7 | DDC 572/.696--dc23
LC record available at https://lccn.loc.gov/2016053681

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Contents
Series Preface............................................................................................................vii
Preface........................................................................................................................ix
Contributors...............................................................................................................xi

Chapter 1 Electrophysiological Methods for Recording CRAC and

TRPV5/6 Channels................................................................................1
J. Ashot Kozak and Grigori Rychkov

Chapter 2 Studies of Structure–Function and Subunit Composition of

Orai/STIM Channel.............................................................................25
Marc Fahrner, Rainer Schindl, and Christoph Romanin

Chapter 3 Signaling ER Store Depletion to Plasma Membrane Orai Channels...... 51

Aparna Gudlur and Patrick Hogan

Chapter 4 Modulation of Orai1 and STIM1 by Cellular Factors.........................73

Jin Seok Woo, Sonal Srikanth, and Yousang Gwack

Chapter 5 CRAC Channels and Ca2+-Dependent Gene Expression.....................93

Yi-Chun Yeh and Anant B. Parekh

Chapter 6 Function of Orai/Stim Proteins Studied in Transgenic Animal

Models...............................................................................................107
Masatsugu Oh-Hora and Xiuyuan Lu

Chapter 7 Assessing the Molecular Nature of the STIM1/Orai1 Coupling

Interface Using FRET Approaches....................................................127
Yandong Zhou, Youjun Wang, and Donald L. Gill

Chapter 8 Optogenetic Approaches to Control Calcium Entry in

Non-Excitable Cells..........................................................................145
Lian He, Qian Zhang, Yubin Zhou, and Yun Huang

Chapter 9 Regulation of Orai/STIM Channels by pH........................................161

Albert S. Yu, Zhichao Yue, Jianlin Feng, and Lixia Yue

v
vi Contents

Chapter 10 Non-Orai Partners of STIM Proteins: Role in ER–PM

Communication and Ca2+ Signaling..................................................177
Klaus Groschner, Niroj Shrestha, and Nicola Fameli

Chapter 11 Store-Independent Orai Channels Regulated by STIM.....................197

Xuexin Zhang, Maxime Gueguinou, and Mohamed Trebak

Chapter 12 Regulation and Role of Store-Operated Ca2+ Entry in Cellular

Proliferation.......................................................................................215
Rawad Hodeify, Fang Yu, Raphael Courjaret, Nancy Nader,
Maya Dib, Lu Sun, Ethel Adap, Satanay Hubrack, and
Khaled Machaca

Chapter 13 TRPV5 and TRPV6 Calcium-Selective Channels............................241

Ji-Bin Peng, Yoshiro Suzuki, Gergely Gyimesi, and
Matthias A. Hediger

Chapter 14 Determining the Crystal Structure of TRPV6...................................275

Kei Saotome, Appu K. Singh, and Alexander I. Sobolevsky

Chapter 15 Identifying TRP Channel Subunit Stoichiometry Using

Combined Single Channel Single Molecule Determinations
(SC-SMD).........................................................................................293
Laura G. Ceballos, Alexander Asanov, and Luis Vaca

Chapter 16 Pharmacology of Store-Operated Calcium Entry Channels..............311

Gary S. Bird and James W. Putney, Jr.

Index ��������������������������������������������������������������������������������������������������������������������325
Series Preface
The concept of signal transduction is now long established as a central tenet of
­biological sciences. Since the inception of the field close to 50 years ago, the number
and variety of signal transduction pathways, cascades, and networks have steadily
increased and now constitute what is often regarded as a bewildering array of mecha-
nisms by which cells sense and respond to extracellular and intracellular environ-
mental stimuli. It is not an exaggeration to state that virtually every cell function is
dependent on the detection, amplification, and integration of these signals. Moreover,
there is increasing appreciation that in many disease states, aspects of signal trans-
duction are critically perturbed.
Our knowledge of how information is conveyed and processed through these cel-
lular molecular circuits and biochemical switches has increased enormously in scope
and complexity since this series was initiated 15 years ago. Such advances would
not have been possible without the supplementation of older technologies, drawn
chiefly from cell and molecular biology, biochemistry, physiology, pharmacology,
with newer methods that make use of sophisticated genetic approaches, as well as
structural biology, imaging, bioinformatics, and systems biology analysis.
The overall theme of this series continues to be the presentation of the wealth of
up-to-date research methods applied to the many facets of signal transduction. Each
volume is assembled by one or more editors who are preeminent in their specialty. In
turn, the guiding principle for editors was to recruit chapter authors who can describe
procedures and protocols with which they are intimately familiar in a reader-friendly
format. The intent is to assure that each volume is of maximum practical value to a
broad audience, including students and researchers just entering an area, as well as
seasoned investigators.
It is hoped that the information contained in the books of this series will constitute
a useful resource to the life sciences research community well into the future.

Joseph Eichberg
Michael Xi Zhu
Series Editors

vii
Preface
Virtually every eukaryotic cell expresses at least some type of calcium channel, in the
plasma membrane, in intracellular organelles, or typically both. Calcium channels in
the plasma membrane have long been of interest to biologists because of their vulner-
ability to modulation by extracellular factors, with either pathological or therapeutic
consequences. Plasma membrane calcium channels are regulated by membrane volt-
age or by ligands, and in some cases by both. Not surprisingly, voltage-activated
channels are generally encountered in cells that depend largely on excitable behavior,
for example, muscle and nerve. Calcium channels that are activated by ligands are
more broadly distributed but are the exclusive mediators of transmembrane calcium
flux in non-excitable cells, for example, blood cells and epithelial cells.
The history of voltage-activated calcium channels is nicely summarized in a review
by Richard Tsien [1], dating back to the seminal work of Sidney Ringer at the end of
the nineteenth century. In non-excitable cells, the history is more recent, and much of
the early work was carried out in smooth muscles, a tissue that expresses both voltage-
activated and ligand-gated calcium channels. It might be said that the first mechanis-
tic insights into calcium channels in non-excitable cells came not from studies of the
channels themselves but of the initial receptor mechanisms upstream. By far, the most
commonly encountered receptor signaling mechanism leading to calcium regulation
in non-excitable cells is the receptor-activated phosphatidylinositol 4,5-bisphosphate–
directed phospholipase C. Through distinct downstream mechanisms, this initial enzy-
matic event gives rise to the activation of the two major classes of non-voltage-activated
calcium channels, the store-operated channels and TRPC channels, a subgroup of the
larger TRP ion channel superfamily. The store-operated channels are activated in
response to the depletion of endoplasmic reticulum stores by the phospholipase C prod-
uct, inositol 1,4,5-trisphosphate, while the TRPCs appear to be activated by phospholi-
pase C–mediated changes in membrane lipid composition. And not surprisingly, there
are instances in which the two channels interact and regulate one another. The history
of the phospholipase C signaling field is well described by Robert Michell [2], and the
histories of the TRP channels and store-operated channels are well described by Craig
Montell and Roger Hardie [3,4] and Jim Putney [5], respectively. This volume focuses
on physiologically and potentially clinically important channels in non-excitable cells,
primarily the store-operated channels and TRP channels, including TRPC channels and
the highly Ca2+-selective TRPV5 and TRPV6. In keeping with the theme of this series,
there is considerable emphasis on the specific methods required for studying them.
This topic was addressed in part in an earlier volume in this series published in 1999
focusing more broadly on calcium signaling [6]. An underlying impetus to produce the
current volume comes from the remarkable advances in this field in the past few years.
Following the discovery of the first mammalian TRP channels by Zhu and Birnbaumer,
the superfamily of TRP channels virtually exploded. After years of false leads, the
molecular components of store-operated channels were revealed by the use of modern
high-throughput genetic screens, first the calcium sensor STIM, and subsequently the
pore-forming store-operated channel subunit, Orai.

ix
x Preface

In addition to the in-depth analysis of the molecular and physiological aspects of

these channels, the reader will no doubt be struck by the breadth of methodological
approaches involved in studying them. Enough cannot be said of the contributions of
the late Roger Tsien in developing readily available chemical as well as genetically
encoded calcium indicators, making possible the measurement of calcium concentra-
tion in the cytoplasm and in organelles in real time. Complementing this approach
is the use of the techniques of electrophysiology, FRET, optogenetics, and x-ray
crystallography, to name a few. Hopefully, this collection will be of use to scientists
investigating these important channels, from professor to graduate student, and in
academia, government, or industry.

MATLAB® is a registered trademark of The MathWorks, Inc. For product informa-

tion, please contact:
The MathWorks, Inc.
3 Apple Hill Drive
Natick, MA 01760-2098 USA
Tel: 508-647-7000
Fax: 508-647-7001
E-mail: [email protected]
Web: www.mathworks.com

REFERENCES
1. Tsien, R.W. and Barrett, C.F. 2005. A brief history of calcium channel discovery. In
Voltage Gated Calcium Channels. G. Zamponi, ed. Plenum Press, New York, pp. 27–47.
2. Michell, R.H. 1986. Inositol lipids and their role in receptor function: History and gen-
eral principles. In Phosphoinositides and Receptor Mechanisms. J.W. Putney, ed. Alan
R. Liss, Inc., New York, pp. 1–24.
3. Hardie, R.C. 2011. A brief history of TRP: Commentary and personal perspective.
Pflügers Archiv: European Journal of Physiology 461:493–498.
4. Montell, C. 2011. The history of TRP channels, a commentary and reflection. Pflügers
Archiv: European Journal of Physiology 461:499–506.
5. Putney, J.W. 2007. Recent breakthroughs in the molecular mechanism of capacitative
calcium entry (with thoughts on how we got here). Cell Calcium 42:103–110.
6. Putney, J.W. 1999. Calcium Signaling. CRC Press, Boca Raton, FL.
Contributors
Ethel Adap Nicola Fameli
Department of Physiology and Biophysics Institute of Biophysics
Weill Cornell Medicine Qatar Medical University of Graz
Doha, Qatar Graz, Austria

Alexander Asanov Jianlin Feng

TIRF Labs Inc. Department of Cell Biology
Cary, North Carolina University of Connecticut School
of Medicine
Farmington, Connecticut
Gary S. Bird
Signal Transduction Laboratory Donald L. Gill
National Institute of Environmental Department of Cellular and Molecular
Health Sciences Physiology
National Institutes of Health Pennsylvania State University College
Research Triangle Park, North Carolina of Medicine
Hershey, Pennsylvania
Laura G. Ceballos
Department of Cell and Developmental Klaus Groschner
Biology Institute of Biophysics
Institute of Cellular Physiology Medical University of Graz
National Autonomous University of Graz, Austria
Mexico
Mexico City, Mexico Aparna Gudlur
Division of Signaling and Gene
Raphael Courjaret Expression
Department of Physiology and Biophysics La Jolla Institute for Allergy and
Weill-Cornell Medical College Immunology
Doha, Qatar La Jolla, California

Maxime Gueguinou
Maya Dib Department of Cellular and Molecular
Department of Physiology and Biophysics Physiology
Weill Cornell Medicine Qatar Pennsylvania State University College
Doha, Qatar of Medicine
Hershey, Pennsylvania
Marc Fahrner
Life Science Center Yousang Gwack
Institute of Biophysics Department of Physiology
Johannes Kepler University University of California
Linz, Austria Los Angeles, California

xi
xii Contributors

Gergely Gyimesi Satanay Hubrack

Institute of Biochemistry and Molecular Department of Physiology and Biophysics
Medicine Weill Cornell Medicine Qatar
and Doha, Qatar
Swiss National Center of Competence
in Research J. Ashot Kozak
TransCure Department of Neuroscience, Cell
University of Bern Biology and Physiology
Bern, Switzerland Boonshoft School of Medicine
Wright State University
Lian He Dayton, Ohio
Institute of Biosciences and Technology Xiuyuan Lu
Texas A&M University Health Science Division of Molecular Immunology
Center Medical Institute of Bioregulation
Houston, Texas Kyushu University
Fukuoka, Japan
Matthias A. Hediger
Institute of Biochemistry and Molecular Khaled Machaca
Medicine Department of Physiology and Biophysics
and Weill Cornell Medicine Qatar
Swiss National Center of Competence Doha, Qatar
in Research
TransCure Nancy Nader
University of Bern Department of Physiology and Biophysics
Bern, Switzerland Weill Cornell Medicine Qatar
Doha, Qatar
Rawad Hodeify Masatsugu Oh-Hora
Department of Physiology and Biophysics Division of Molecular Immunology
Weill Cornell Medicine Qatar Medical Institute of Bioregulation
Doha, Qatar Kyushu University
Fukuoka, Japan
Patrick Hogan
Division of Signaling and Gene Anant B. Parekh
Expression Department of Physiology, Anatomy
La Jolla Institute for Allergy and and Genetics
Immunology University of Oxford
La Jolla, California Oxford, United Kingdom
Ji-Bin Peng
Yun Huang Division of Nephrology
Center for Epigenetics and Disease Nephrology Research and Training Center
Prevention and
Texas A&M University Health Science Department of Urology
Center University of Alabama at Birmingham
Houston, Texas Birmingham, Alabama
Contributors xiii

James W. Putney, Jr. Appu K. Singh

Signal Transduction Laboratory Department of Biochemistry and
National Institute of Environmental Molecular Biophysics
Health Sciences Columbia University
National Institutes of Health New York, New York
Research Triangle Park, North Carolina
Alexander I. Sobolevsky
Christoph Romanin Department of Biochemistry and
Life Science Center Molecular Biophysics
Institute of Biophysics Columbia University
Johannes Kepler University New York, New York
Linz, Austria
Sonal Srikanth
Grigori Rychkov Department of Physiology
School of Medicine University of California
University of Adelaide Los Angeles, California
and
South Australian Health and Medical Lu Sun
Research Institute Department of Physiology and Biophysics
Adelaide, South Australia, Australia Weill Cornell Medicine Qatar
Doha, Qatar
Kei Saotome
Department of Neuroscience Yoshiro Suzuki
Howard Hughes Medical Institute Division of Cell Signaling
Dorris Neuroscience Center National Institutes of Natural Sciences
The Scripps Research Institute and
La Jolla, California Department of Physiological Sciences
SOKENDAI (The Graduate University
and for Advanced Studies)
Okazaki, Japan
Department of Integrative Structural
and Computational Biology Mohamed Trebak
The Scripps Research Institute Department of Cellular and Molecular
La Jolla, California Physiology
Pennsylvania State University College
Rainer Schindl of Medicine
Life Science Center Hershey, Pennsylvania
Institute of Biophysics
Johannes Kepler University Luis Vaca
Linz, Austria Department of Cell and Developmental
Biology
Niroj Shrestha Institute of Cellular Physiology
Institute of Biophysics National Autonomous University of
Medical University of Graz Mexico
Graz, Austria Mexico City, Mexico
xiv Contributors

Youjun Wang Zhichao Yue

Beijing Key Laboratory of Gene Department of Cell Biology
Resources and Molecular University of Connecticut School
Development of Medicine
Beijing Normal University Farmington, Connecticut
Haidian, Beijing, People’s Republic
of China Qian Zhang
Institute of Biosciences and Technology
Jin Seok Woo Texas A&M University Health Science
Department of Physiology Center
University of California Houston, Texas
Los Angeles, California Xuexin Zhang
Department of Cellular and Molecular
Yi-Chun Yeh Physiology
Department of Physiology, Anatomy Pennsylvania State University College
and Genetics of Medicine
University of Oxford Hershey, Pennsylvania
Oxford, United Kingdom
Yandong Zhou
Albert S. Yu Department of Cellular and Molecular
Department of Cell Biology Physiology
University of Connecticut School Pennsylvania State University College
of Medicine of Medicine
Farmington, Connecticut Hershey, Pennsylvania

Yubin Zhou
Fang Yu Institute of Biosciences and Technology
Department of Physiology and Biophysics Texas A&M University Health Science
Weill Cornell Medicine Qatar Center
Doha, Qatar Houston, Texas
and
Lixia Yue
Department of Cell Biology Department of Medical Physiology
University of Connecticut School Texas A&M University Health Science
of Medicine Center
Farmington, Connecticut Temple, Texas
 1 Electrophysiological
Methods for Recording
CRAC and TRPV5/6
Channels
J. Ashot Kozak and Grigori Rychkov

CONTENTS
1.1 Introduction........................................................................................................1
1.2 Characteristics of Calcium Entry in Non-Excitable Cells.................................2
1.3 CRAC Channels.................................................................................................4
1.3.1 CRAC Channels in the Native Environment.........................................4
1.3.2 CRAC Current–Voltage Relation...........................................................5
1.3.3 Current Separation.................................................................................5
1.3.4 Perforated-Patch Recording...................................................................7
1.3.5 CRAC Channel Activity with Various Permeating Cations...................7
1.3.6 CRAC Single-Channel Conductance.....................................................8
1.3.7 Heterologously Expressed Orai/STIM Channels.................................11
1.4 TRPV5 and TRPV6 Channels.........................................................................14
1.4.1 Heterologously Expressed TRPV5/6...................................................14
1.4.2 Endogenous TRPV5/6 Channels.........................................................15
1.4.3 Single-Channel Conductance...............................................................16
Acknowledgment......................................................................................................16
References.................................................................................................................16

1.1 INTRODUCTION
During the past two decades, great advances have been made in the electrophysio-
logical and molecular identification of calcium entry pathways in non-excitable cells.
The term “non-excitable” refers to a variety of cell types that are not capable of firing
action potentials. Essentially, except for neurons, muscle cells, and some endocrine
cells, all other cells in the body are non-excitable. For the most part, they lack the
necessary levels of expression of voltage-gated Na+ channels (NaV family) and also
voltage-gated Ca2+ channels (CaV family). Ca2+ influx in these cell types is therefore
thought to rely on unrelated, voltage-independent channels, such as Orai (CRACM)
and TRP family members. In this chapter, we will discuss direct electrophysiological
methods used to record the electrical activity of these proteins, focusing on calcium-
selective Orai/STIM and TRPV5/TRPV6 channels.

1
2 Calcium Entry Channels in Non-Excitable Cells

1.2 CHARACTERISTICS OF CALCIUM ENTRY

IN NON-EXCITABLE CELLS
One of the first non-excitable cell types used to investigate the function of non-­voltage-
gated Ca2+ channels was cells of the immune system [1]. In T lymphocytes and mast
cells, store-operated calcium entry is the main pathway providing cytoplasmic cal-
cium elevations necessary for key cellular functions, such as antigenic activation,
proliferation, and degranulation. Calcium stores inside the cell that were shown to be
important for CRAC channel activation and functions are the endoplasmic reticulum
(ER) and mitochondria [2–6].
Direct evidence for calcium entry following store depletion was demonstrated
using the now classical calcium readdition protocol in cells loaded with calcium
indicator dyes, such as Fura-2. Figure 1.1 shows a calcium imaging experiment in
Jurkat T lymphocytes loaded with Fura-2 ratiometric calcium dye. Upon the removal
of calcium and the simultaneous addition of sarcoplasmic/endoplasmic reticulum
calcium ATPase (SERCA) pump inhibitor cyclopiazonic acid (CPA) [7], a transient
calcium elevation was observed. This is believed to represent the release of ion-
ized calcium from the ER into the cytoplasm (Fura-2 indicator is in the cytoplasm).
Calcium release in this case is mediated through a “Ca2+ leak” pathway operating in
the ER membrane. Normally, SERCA acts to sequester cytoplasmic calcium into the
ER, counteracting this outwardly directed calcium leak. When SERCA is blocked by

CPA
2 Ca, 4 NaCl
2 Ca
1 EGTA, 0 Ca 140 NaCl/4 NaCl
4
2 Ca 2 Ca, 140 NaCl 140 KCl
Mean ratio (340 nm/380 nm)

Mean ratio (340 nm/380 nm)

140 NaCl
4 140 NaCl 4 KCl/140 KCl 4 4 KCl
4 KCl
4 KCl
3
3 4 KCl 3

2
2 2

140 KCl
1 1 1
0 5 10 15 20 0 5 10
(a) Time (min) (b) Time (min)

FIGURE 1.1 Fura-2 measurement of cytosolic calcium in intact human Jurkat T lympho-
cytes (a) and murine MIN6 β cells (b). (a) Calcium entry through endogenous Orai channels
was measured with the calcium readdition protocol after ER store depletion with 20 μM CPA.
External [K+] was either 4 mM (shown in black) or elevated to 140 mM (in red) after calcium
was reintroduced to the bath. [Na+] was 140 and 4 mM for (a) and (b), respectively. What, if
any, influence the concomitant changes in bathing [Na+] have on SOCE is not known. Each
black and red symbol represents the mean response from 49 and 36 cells. (b) Calcium entry
through endogenous CaV channels [142] was evoked by [K+] elevation in the bathing solution
(as in (a)). Cl− concentration was kept constant in (a) and (b). Each symbol represents the mean
response from 14 cells. Error bars in (a) and (b) represent SEM.
Electrophysiological Methods for Recording CRAC and TRPV5/6 Channels 3

CPA, however, the calcium flux into the cytoplasm due to the leak pathway is no lon-
ger counteracted and this is manifested as a calcium elevation. The transient nature of
this elevation is likely due to plasma membrane Ca2+ ATPase (PMCA), which is not
sensitive to CPA and can still expel calcium ions from the cytoplasm.
The basal ER “Ca2+ leak” pathway remains an enigma both in terms of its molecu-
lar identity and the factors that regulate it. It is thought to participate in determining
the ER calcium content (also termed “calcium load”). Over the years several ion
channels have been proposed to underlie the ER leak such as the translocon complex
(translocation channel), pannexins, TRPP2 (polycystin), Bcl2 anti-apoptotic pro-
teins, and even IP3R channels functioning in the absence of ligand binding [8–12].
It is presently unclear if Ca2+ leak channels are regulated by ER calcium content and
cytoplasmic Ca2+ or if they are constitutively open. In whole-cell patch-clamp experi-
ments, the leak pathway appears to function throughout the duration of the recording,
enabling the prolonged continuous detection of CRAC channel activity with passive
store depletion. Almost no information is available on their pharmacology. Basal
leak pathways have also been described in the cardiac sarcoplasmic reticulum, and
ryanodine receptors have been suggested to participate in basal leak under specific
circumstances [10].
The reintroduction of calcium to the bathing solution (in the presence of CPA)
results in a large calcium elevation, which represents store-operated calcium entry
(SOCE). This process is a direct consequence of emptying the ER calcium store,
as this pathway is not active without CPA or thapsigargin application. In lympho-
cytes, SOCE is usually larger than the ER release transient and also decays more
slowly. When extracellular [K+] is increased from 4 to 140 mM (Figure 1.1b) cal-
cium increases are drastically diminished. K+ elevation moves the potassium Nernst
potential from −90 mV to approximately 0 mV, a 90 mV depolarization. This is
expected to depolarize the membrane potential of the Jurkat T cell, which is set
by the voltage-gated KV1.3 and calcium-activated KCa3.1 channels, as well as the
two-pore voltage-independent K+ channels [13,14]. Depolarized potentials result
in a reduced driving force for Ca2+, thereby decreasing Ca2+ influx. This in turn
reduces KCa3.1 currents in a positive feedback loop, causing Ca2+ transients to
decay faster [1]. Importantly, the shape (time course) of SOCE is set by PMCA
activity [6,15–18]. In short, membrane depolarization reduces rather than increases
SOCE, demonstrating that in lymphocytes, voltage-gated CaV channels are not the
underlying calcium influx pathway. Accordingly, the blockade of KV1.3 in lympho-
cytes invariably inhibits SOCE and suppresses proliferation, which requires SOCE
[19,20]. Similar SOCE-K+ channel systems exist in other cell types [21]. Note that
the CPA-induced store calcium transient is not affected by the rise in the extracel-
lular K+ concentration since it does not depend on the plasma membrane potential.
SOCE is not entirely abolished in high K+, presumably because the calcium equi-
librium potential is well above 0 mV [22]. By contrast, in excitable cells, such as
pancreatic β cells, depolarizations caused by increasing [K+] result in a substantial
Ca2+ influx through voltage-gated Ca2+ channels of the CaV family (Figure 1.1b).
In β cells the major CaV subtype is the dihydropyridine-sensitive L-type channel,
which opens at membrane potentials above −10 mV [23,24].
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