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Preface
This book is about how chloride ions are regulated neurotransmitter uptake, and cell volume control.
and how they cross the plasma membrane of neurons, Disruption of chloride homeostasis in neurons, glial
glial and epithelial cells. It spans from the molecu- and epithelial cells underlies diverse pathological
lar structure and function of carriers and channels conditions such as epilepsy, deafness, imbalance,
involved in chloride transport to their role in vari- brain edema, and neurogenic inflammation. Further,
ous neurological diseases. The importance of chloride primary brain tumor cells (e.g. glioma cells) migrate
ions in cell physiology has not been fully recognized within the brain utilizing chloride channels and
until recently. This is in spite of the fact that chloride, transporters expressed in their plasma membrane.
in addition to bicarbonate, is the most abundant free Accordingly, drugs that target chloride channels (e.g.
anion in animal cells, and performs or determines chlorotoxin) are being studied as possible therapeutic
fundamental biological functions in all tissues. For agents in primary brain tumor growth and invasion.
many years it was thought that chloride was distrib- Further, drugs that block cation-coupled-chloride
uted in thermodynamic equilibrium across the plasma cotransporters, such as 5-sulfamoyl benzoic acid
membrane of most cells. It took several decades to derivatives (e.g. bumetanide), that have been used for
eradicate this erroneous view that had become a text- many years as diuretics, are now being investigated
book dogma. This is probably one of the reasons why as potential neuroprotective compounds in brain
it has taken such a long time to begin to recognize ischemic damage, as therapeutic agents in neonatal
the importance of chloride ions in neuronal function epileptic seizures, and as possible analgesic and anti-
and dysfunction, compared with the weight given to inflammatory agents in acute tissue injury.
the cations sodium, calcium and potassium. Research This book brings together most of the primary
carried out during the last couple of decades has research information about the molecular physiol-
led to a dramatic change in this simplistic view. We ogy and pathology of chloride carriers and channels
now know that most animal cells, neurons included, scattered throughout the scientific literature. The
exhibit a non-equilibrium distribution of chloride forerunner of the present book, “Chloride Channels
across their plasma membranes. Thus, far from being and Carriers in Nerve and Muscle, and Glial Cells”
passively distributed across the plasma membrane in (Alvarez-Leefmans, F.J. and Russell, J.M. Eds., Plenum,
accordance with the membrane potential, chloride is New York, 1990), appeared prior to the molecular
actively transported and tightly regulated in virtu- biology era. Since its publication a virtual explosion
ally all animal cells. The level of intracellular chloride in our understanding of these transport protein mol-
results from a delicate functional balance between ecules has occurred. However, until now no book or
chloride channels and carriers present in the plasma monograph has effectively integrated the emerging
membrane. Alterations in this balance underlie vari- concepts in a way that is useful to both general and
ous nervous system dysfunctions. specialized neuroscience communities. To address this
Over the last 10 to 15 years, with the spectacu- shortcoming the present book has been assembled
lar growth of molecular biology and the advent of with contributions by world leaders in the field. Our
new optical and electrophysiological methods, an goal has been to create a book that makes available
enormous amount of exciting new information has to both specialists and non-specialists detailed infor-
become available on the molecular structure and mation and fundamental concepts being developed
function of chloride channels and carriers and their in this exponentially expanding field. The contribu-
involvement in various diseases. In nerve cells, chlo- tors present their topics of study not just by review-
ride channels and carriers play key functional roles in ing their own findings but also by putting them in
GABA- and glycine-mediated synaptic signaling in perspective so that others wishing to enter this excit-
the de- and hyperpolarizing directions, including pre- ing field of neuroscience may quickly see the obvious
and postsynaptic inhibition. They also play a central areas where more work is needed.
role in neuronal growth and development, extracel- The book is divided into five parts and thirty chap-
lular potassium scavenging, regulation of intracellu- ters. Many chapters discuss their topics from a histori-
lar pH, sensory-transduction including nociception, cal perspective. This interesting exercise shows that
ix
x Preface
many current concepts were proposed or discovered we have respected the views expressed by the authors
long ago. These basic concepts were the result of con- although they do not always reflect our own.
siderable solid work and insights into cellular physi- Needless to say, this volume is the result of a col-
ology without which current molecular data would lective effort. The editors are particularly grateful to
be meaningless. Extensive introductory sections cover the contributing authors, and to Dr. Johannes Menzel,
basic thermodynamic and kinetics aspects of chloride Ms. Clare Caruana, Ms. Deena Burgess and Ms. Kim
transport, as well as current methods for studying Lander at Elsevier for their support throughout the
chloride regulation, from fluorescent dyes in single editing and production process. We would also like
cells to knock-out models. to express our thanks to our respective institutions,
All chapters have been carefully edited and cross- Wright State University Boonshoft School of Medicine,
referenced to avoid unnecessary repetition and to and Vanderbilt University School of Medicine, for
provide the reader with easy referencing to sections their continual support of the work dedicated to put-
of the book where related information can be found. ting together the present volume.
We have strived to attain continuity between the
chapters and have emphasized the use of a unified Francisco Javier Alvarez-Leefmans
nomenclature. Nevertheless, as editors, not censors, Eric Delpire
List of Contributors
Norma C. Adragna Department of Pharmacology Amal K. Dutta Department of Cell Physiology,
and Toxicology and Cell Biophysics Group, Boonshoft National Institute for Physiological Sciences, Okazaki,
School of Medicine, Wright State University, Dayton, Japan
OH, USA
J. Clive Ellory Department of Physiology, Ana-
Norio Akaike Research Division for Life Sciences, tomy & Genetics, University of Oxford, Oxford, UK
Kumamoto Health Science University, Kumamoto, Japan
Stephan Frings Department of Molecular Physio-
Francisco Javier Alvarez-Leefmans Department logy, University of Heidelberg, Heidelberg, Germany
of Pharmacology & Toxicology, Boonshoft School of
Medicine, Wright State University, Dayton, OH, USA Kenneth Gagnon Departments of Anesthesiology
and Radiation Oncology, Vanderbilt University School
George J. Augustine Department of Neurobiology, of Medicine, Nashville, TN, USA
Duke University Medical Center, Durham, NC, USA;
Laboratory of Synaptic Circuitry, Duke-NUS Graduate Gerardo Gamba Molecular Physiology Unit,
Medical School, Singapore; A*STAR/Duke-NUS Instituto Nacional de Ciencias Médicas y Nutrición
Neuroscience Research Partnership, Singapore, and Salvador Zubirán and Instituto de Investigaciones
Department of Physiology, National University of Biomédicas, Universidad Nacional Autónoma de
Singapore, Singapore. México, Mexico City, Mexico
Ken Berglund Department of Neurobiology, Duke Nicole Garbarini Department of Anesthesiology,
University Medical Center, Durham, NC, USA Vanderbilt University School of Medicine, Nashville,
TN, USA
Emmanuel J. Botzolakis The Medical Scientist
Training Program, Vanderbilt University School of John S. Gibson Department of Veterinary
Medicine, Nashville, TN, USA Medicine, University of Cambridge, Cambridge, UK
Fiona C. Britton Department of Physiology & Cell Steffen Hamann Nordic Centre for Water
Biology, University of Nevada School of Medicine, Imbalance Related Disorders, Institute of Cellular and
Reno, NV, USA Molecular Medicine, The Panum Institute, University
of Copenhagen, Denmark
Peter D. Brown Faculty of Life Sciences, University
of Manchester, Manchester, UK H. Criss Hartzell Department of Cell Biology and
Center for Neurodegenerative Disease, Emory Univer-
Min-Hwang Chang Physiology & Biomedical
sity School of Medicine, Atlanta, GA,USA
Engineering, Mayo Clinic College of Medicine,
Rochester, MN, USA Hana Inoue Department of Cell Physiology,
National Institute for Physiological Sciences, Okazaki,
Sarah L. Davies Faculty of Life Sciences, University
Japan
of Manchester, Manchester, UK
Yves De Koninck Department of Psychiatry, Thomas J. Jentsch Leibniz-Institut für Molekulare
Division of Cellular Neurobiology, Centre de recherche Pharmakologie (FMP) and Max-Delbrück-Centrum
université Laval Robert-Giffard, Québec, QC, Canada für Molekulare Medizin (MDC), Berlin, Germany
Eric Delpire Departments of Anesthesiology Kristopher T. Kahle Department of Neurosurgery,
and Molecular Physiology & Biophysics, Vanderbilt Massachusetts General Hospital and Harvard Medical
University School of Medicine, Nashville, TN, USA School, Boston, MA, USA
Mauricio Di Fulvio Department of Pharmacology James L. Kenyon Departments of Physiology &
and Toxicology, Boonshoft School of Medicine, Wright Cell Biology and Pharmacology, University of Nevada
State University, Dayton, OH, USA School of Medicine, Reno, NV, USA
xi
xii List of Contributors
Douglas B. Kintner Department of Neurological John M. Russell Department of Biology, Life
Surgery, University of Wisconsin School of Medicine Sciences Complex, Syracuse University, Syracuse,
and Public Health, Madison, WI, USA NY, USA
Thomas Kuner Institute of Anatomy and Cell Bio- Ravshan Z. Sabirov Laboratory of Molecular
logy, University of Heidelberg, Heidelberg, Germany Physiology, Institute of Physiology and Biophysics,
Peter K. Lauf Cell Biophysics Group, Boonshoft Tashkent, Uzbekistan; and Department of Cell
School of Medicine, Wright State University, Dayton, Physiology, National Institute for Physiological
OH, USA Sciences, Okazaki, Japan.
Normand Leblanc Department of Pharmacology, Kaori Sato Department of Cell Physiology, National
University of Nevada School of Medicine, Reno, NV, Institute for Physiological Sciences, Okazaki, Japan
USA
Harald Sontheimer Department of Neurobiology
Nanna MacAulay Nordic Centre for Water and Center for Glial Biology in Medicine, University
Imbalance Related Disorders, Institute of Cellular and of Alabama, Birmingham, AL, USA
Molecular Medicine, The Panum Institute, University
Kevin J. Staley Division of Pediatric Neurology,
of Copenhagen, Denmark
Massachusetts General Hospital, and Harvard Medical
Robert L. Macdonald Departments of Neurology,
School Boston, MA, USA
Pharmacology, and Molecular Physiology &
Biophysics, Vanderbilt University School of Medicine, Tobias Stauber Leibniz-Institut für Molekulare
Nashville, TN, USA Pharmakologie (FMP) and Max-Delbrück-Centrum
für Molekulare Medizin (MDC), Berlin, Germany
Daniel C. Marcus Cellular Biophysics Laboratory,
Department of Anatomy & Physiology, Kansas State Dandan Sun Department of Neurological Surgery,
University, Manhattan, KS, USA University of Wisconsin School of Medicine and Public
Health, Madison, WI, USA
Ian D. Millar Faculty of Life Sciences, University
of Manchester, Manchester, UK Makoto Suzuki Edogawabashi Suzuki Clinic,
David Mount Renal Division, Brigham and Tokyo, Japan
Women’s Hospital, Harvard Institutes of Medicine,
Abduqodir H. Toychiev Department of Cell Phy-
Boston MA, USA
siology, National Institute for Physiological Sciences,
Gaia Novarino Leibniz-Institut für Molekulare Okazaki, Japan
Pharmakologie (FMP) and Max-Delbrück-Centrum
Noga Vardi Department of Neuroscience, School
für Molekulare Medizin (MDC), Berlin, Germany
of Medicine, University of Pennsylvania, Philadelphia,
Martha O’Donnell Department of Physiology and PA, USA
Membrane Biology, School of Medicine, University of
Alan S. Verkman Departments of Medicine and
California, Davis, CA, USA
Physiology, Cardiovascular Research Institute, Uni-
Yasunobu Okada Department of Cell Physiology,
versity of California, San Francisco, CA, USA
National Institute for Physiological Sciences, Okazaki,
Japan Philine Wangemann Cellular Physiology Labora-
tory, Department of Anatomy & Physiology, Kansas
John A. Payne Department of Physiology and
State University, Manhattan, KS, USA
Membrane Biology, School of Medicine, University of
California, Davis, CA, USA Thomas Zeuthen Nordic Centre for Water
Imbalance Related Disorders, Institute of Cellular and
Brooks B. Pond Department of Pharmaceutical
Molecular Medicine, The Panum Institute, University
Sciences, East Tennessee State University College of
of Copenhagen, Denmark
Pharmacy, Johnson City, TN, USA
Michael F. Romero Physiology & Biomedical Ling-Li Zhang Department of Neuroscience,
Engineering, and Nephrology & Hypertension, Mayo School of Medicine, University of Pennsylvania,
Clinic College of Medicine, Rochester, MN, USA Philadelphia, PA, USA
P A R T I
Overview Of chlOride
transpOrters and channels
C H A P T E R
1
Chloride Channels: An Historical Perspective
H. Criss Hartzell
o u T l I n E
I. Introduction VII. Structure and Function 8
II. Chloride ‘‘Passivity” VIII. Diseases Caused by Disorders of Cl
Channels and Transporters 9
III. Active Chloride Transport 4
IV. Technical Hurdles to Studying Cl Channels 5 IX. Intracellular Cl Channels 9
V. The Chloride Awakening 6 X. Chloride may Regulate Protein Function 10
A. Single Channel Properties of ClC-0 6 XI. Other Functions of Cl Channels 11
B. Myotonia Congenita 6
C. Cystic Fibrosis 7 XII. Concluding Remarks 11
VI. Cl Channel Genes 7 References 12
I. InTRODuCTIOn scholarly, and balanced approaches, the reader should
consult a number of reviews on Cl channels (Nilius
et al., 1997; Hume et al., 2000; Nilius and Droogmans,
One reason I became a scientist was that other disci-
2001; Welsch et al., 2001; Eggermont et al., 2001; Jentsch
plines, such as history, frightened me because their reali-
et al., 2002; Nilius and Droogmans, 2003; Faundez and
ties seemed variable, dictated by perception rather than
Hartzell, 2004; Hartzell et al., 2005; Sile et al., 2006;
by hard data. Although I have learned that science, too,
Okada et al., 2006; Puljak and Kilic, 2006; Gadsby
is encumbered by perceptual distortions, it was never-
et al., 2006; Zifarelli and Pusch, 2007; Hartzell et al., 2008;
theless unsettling for me to accept an invitation to write
Jentsch, 2008) as well as Chapters 12–15 in this book.
a chapter with ‘‘history’’ in the title. I simply do not trust
my historical perceptions. So, at the outset, I apologize
deeply to those I have misrepresented or forgotten. This
II. CHlORIDe ‘‘PASSIVITy”
chapter is not meant to be a comprehensive history of
Cl channels, but rather is meant to present my personal
view of how Cl channels have finally begun to attract Most ion channel specialists will agree, I think, that
the attention they deserve. For more comprehensive, our understanding of how anion channels work still
Physiology and Pathology of Chloride Transporters and Channels in the Nervous System © 2009, Elsevier Inc.
4 1. CHloRIdE CHAnnEls: An HIsToRICAl PERsPECTIvE
lags significantly behind our understanding of cation Nevertheless, at the time, with skeletal muscle,
channels. In Bertil Hille’s first edition of the ‘‘bible’’ erythrocytes, and squid axon all weighing in on the
on ion channels published in 1984 (Hille, 1984), fewer side of Cl being in electrochemical equilibrium,
than three pages were devoted to Cl channels. Why other data to the contrary seemed not to receive much
was this? Certainly, there is a natural tendency to attention. Results from a number of tissues did not fit
embrace positivity over negativity, but the reason is into the equilibrium model. Contradicting the data of
certainly deeper. As a graduate student in the late Steinbach, Richard D. Keynes (Keynes, 1963) showed
1960s, I remember learning that Cl channels did not clearly that intracellular Cl concentration in squid
exist. Actually, it is highly unlikely that such a state- axon was 2–3 times higher than equilibrium and that
ment was ever made by my professors, but it is how active Cl uptake must occur to explain this. But, to
my naïve mind heard it. Probably I was told that Cl the public, perhaps a two-fold gradient seemed unim-
moved through membranes by a purely electro-diffu- portant compared to the 10- to 1000-fold gradients
sive mechanism and that Cl was distributed passively that cations normally exhibited. Strong evidence also
across the plasma membrane. To me, the statement that existed that Cl was actively transported in gastric
Cl was both ‘‘passive’’ and ‘‘negative’’ had the same and intestinal epithelia (Hogben, 1959), but the idea
effect as the kiss of death. If Cl was passively distrib- that the stomach secreted 1-N HCl may have seemed
uted, it seemed useless: it could not provide energy for bizarre enough in itself to bar Cl from entering the
transport of other substances and its passive distribu- mainstream by this route.
tion precluded a signaling function. But clearly, I was The discovery that GABA hyperpolarized neurons
way off-base in thinking that Cl channels did not exist by opening ligand-gated Cl channels (Kuffler and
just because Cl seemed to be distributed passively. It Edwards, 1958; Boistel and Fatt, 1958) showed that
was well known that muscle, neurons, and other cell E was not equal to resting E in neurons. However,
Cl m
types exhibited Cl conductances. And Hodgkin and the measured reversal potentials of GABA-produced
Horowicz (Hodgkin and Horowicz, 1959) referred to i.p.s.p.’s were so close to the resting potential that is
the Cl permeation pathway they studied in skeletal was concluded that ‘‘normally Cl ions are in electro-
muscle as ‘‘the Cl channel’’, which we now know is chemical equilibrium across the membrane’’ (Coombs
ClC-1 (see Chapter 12, this volume). et al., 1955). Later, it was recognized that GABA pro-
This view of Cl ‘‘passivity’’ came about because, duced depolarizing responses in some neurons, like
at the time, ideas about Cl were dominated by work dorsal root ganglion (De Groat et al., 1972), and it
on skeletal muscle and erythrocytes. In resting skel- became clear that intracellular Cl concentration could
etal muscle, Cl permeability is extremely high: P is change, and therefore Cl must be actively trans-
Cl
more than twice that of P . Because of this high per- ported, at least under certain conditions, as discussed
K
meability, Cl distributes passively according to the throughout this book and specifically in Chapters 2, 5,
membrane potential (Hodgkin and Horowicz, 1959; 17 and 22.
Hutter and Noble, 1960; Adrian, 1960, 1961) and any
active transport of Cl would need to work very hard
to counteract this high permeability. In erythrocytes
III. ACTIVe CHlORIDe TRAnSPORT
the intracellular Cl concentration is also close to
electrochemical equilibrium due to a large Cl con-
ductance and to Cl/HCO exchange. The prevalent Along with the realization that the electrochemical
3
perception, at least in textbooks (e.g. Ruch and Patton, equilibrium of Cl (E E ) in skeletal muscle and
Cl m
1965; Hille, 1984), that Cl was distributed passively erythrocytes was due to their high Cl permeability
in most cells was also bolstered by earlier experiments came the discovery that in cells with low resting Cl
supporting this idea in squid axon (Steinbach, 1941). It permeability, active Cl transport mechanisms were
is important to note, however, that the apparent pas- present and often generated electrochemical Cl gradi-
sive distribution of Cl did not rule out active trans- ents (E E or E E ), as illustrated in Fig. 1.1.
Cl m Cl m
port of Cl, it only meant that the passive leak was Some cells, notably epithelial cells, immature neurons,
much greater than any active transport that might and mature sensory neurons, express transporters
occur. It now is clear that although Cl may appear such as the Na-K-2Cl cotransporter (NKCC), the
to be in electrochemical equilibrium, Cl is actively Cl/HCO exchangers, or the Na-Cl cotransporter
3
transported in muscle as well as other cells. For exam- (NCC) that typically accumulate Cl at concentra-
ple, blocking muscle Cl channels with 9AC reveals tions above electrochemical equilibrium inside the cell.
the presence of active Cl transport (Aickin, 1990; Indeed, the accumulation of intracellular Cl above
Alvarez-Leefmans, 2001; Gosmanov et al., 2003). electrochemical equilibrium is the basis for Cl-driven
Iv. TECHnICAl HuRdlEs To sTudyIng Cl CHAnnEls 5
concentrations of Cl in different cell types can vary
epithelial cells
skeletal muscle
immature neurons mature neurons over nearly an order of magnitude (see Fig. 13.3 in
red blood cells
sensory neurons Chapter 13). Also, although the concentration of cat-
ions, most notably Ca2, can change transiently, these
Cl loaders Cl extruders
NKCC,NCC,AE1 KCC,NDAE changes are generally fast and local. It seems, however,
that changes in [Cl] occur over a slower time scale.
i
Incidentally, this may be another reason why Cl has
been neglected: if one is looking for changes on the
Cl− High [Cl−] Low [Cl−] time scale of seconds, things that take hours to hap-
pen will be missed. It has already been mentioned that
Cl concentration in neurons varies during develop-
ment (see also Chapters 7 and 19 in this volume), but
it also may change dramatically in response to synap-
tic activity (Kuner and Augustine, 2000; Isomura et al.,
Cl is in equilibrium Cl flows outward Cl flows inward 2003; Berglund et al., 2006 and 2008), as discussed in
E = V E > V E < V Chapter 7 in this volume.
Cl m Cl m Cl m
FIGuRe 1.1 Control of cytosolic Cl in different cell types. In
skeletal muscle and erythrocytes, Cl appears to be in electrochemi-
cal equilibrium because there is a large resting Cl conductance IV. TeCHnICAl HuRDleS TO
that masks active Cl transport. In epithelial cells, immature neu-
rons, and adult sensory neurons, Cl loaders establish a higher than STuDyInG Cl CHAnnelS
electrochemical equilibrium intracellular Cl concentration. In most
mature central neurons, Cl extruders establish a lower than elec-
trochemical equilibrium intracellular Cl concentration. There are also technical reasons why the study and
understanding of Cl channels and transporters have
fluid secretion by epithelial cells. In secretory epithe- lagged behind that of cation channels. One reason is
lial cells, basolateral NKCC accumulates Cl in the cell that the pharmacology of Cl channels was, and still
above electrochemical equilibrium. Then, opening of is, distressingly inadequate. Unlike many cation chan-
Cl channels in the apical membrane results in efflux of nels that have very specific drugs that can be used to
Cl down its electrochemical gradient into the lumen, block them (like TTX for voltage-gated Na channels,
followed by paracellular fluxes of Na and water. charybdotoxin for large-conductance K channels and
Other cell types, such as many mature neurons, express conotoxins for voltage-gated Ca2 channels), Cl chan-
transporters that tend to extrude Cl, like the K-Cl nel blockers are notorious for their low affinity and low
cotransporters (KCCs) discussed in Chapter 17 and the specificity. Until recently, the Cl channel pharmaco-
Na-dependent anion exchangers (NDAE) discussed peia has consisted of a selection of relatively low-affin-
in Chapter 4. The resulting lower than electrochemi- ity dirty molecules, including the stilbene disulfonate
cal equilibrium intracellular Cl concentration is the derivatives such as the amino reactive agents DIDS and
basis for the typical hyperpolarizing action of GABA SITS, the diphenylamine-2-carboxylate (DPC) deriva-
in mature neurons, for example. Indeed, in immature tives such as 5-nitro-2-(3-phenylpropylamino) benzoic
neurons that express Cl loaders, and mature primary acid (NPPB) and anthracene-9-carboxylate (9-AC);
sensory neurons, GABA produces depolarizing post- fenamic acids like niflumic acid (NFA) and flufenamic
synaptic potentials, rather than hyperpolarizing ones. acid FFA; and indanyloxyacetic acid 94 (IAA-94). But
The depolarizing responses may be important in sta- these drugs have neither high affinity nor high selectiv-
bilizing synapses during development (Ben-Ari et al., ity. Very recently, there has been progress in identifying
2007) in CNS neurons, and in presynaptic inhibition in extremely high affinity (nanomolar) drugs for some Cl
the central terminals of mature primary sensory neu- channels, notably CFTR and calcium-activated chloride
rons, as discussed in Chapter 22 in this volume. channels (CaCCs) (Yang et al., 2003; Muanprasat et al.,
The recognition that Cl concentration differs in 2004; Verkman et al., 2006; Muanprasat et al., 2007; De
different cell types depending on the complement of La Fuente et al., 2008). Also, peptide toxins for CFTR
expressed Cl transporters has placed Cl in what may and ClC-2 have recently been found (Thompson et al.,
be a special position among biological ions. Although 2005; Fuller et al., 2007). Experience with these toxins is
the concentrations of Na, K, Ca2 and Mg2 can presently limited, but it is likely that these toxins will be
change with cellular activity, the concentrations of extremely useful in studying these channels and opens
these cations under ‘‘resting’’ conditions are very simi- the door to searching for other native toxins directed at
lar in different cell types. In contrast, the intracellular Cl channels (see Chapter 26 in this volume).
