Am J Physiol Renal Physiol
280: F748–F757, 2001.
invited review
Ion permeation and selectivity in ClC-type chloride
channels
CHRISTOPH FAHLKE
Institute of Physiology, RWTH Aachen, 52057 Aachen, Germany,
and Centro de Estudios Cientificos, Valdivia, Chile
Fahlke, Christoph. Ion permeation and selectivity in ClC-type chloride channels. Am J Physiol Renal Physiol 280: F748–F757, 2001.—
Voltage-gated anion channels are present in almost every living cell and
have many physiological functions. Recently, a novel gene family encoding voltage-gated chloride channels, the ClC family, was identified. The
knowledge of primary amino acid sequences has allowed for the study of
these anion channels in heterologous expression systems and made
possible the combination of site-directed mutagenesis and high-resolution electrophysiological measurements as a means of gaining insights
into the molecular basis of channel function. This review focuses on one
particular aspect of chloride channel function, the selective transport of
anions through biological membranes. I will describe recent experiments
using a combination of cellular electrophysiology, molecular genetics,
and recombinant DNA technology to study the molecular basis of ion
permeation and selection in ClC-type chloride channels. These novel
tools have provided new insights into basic mechanisms underlying the
function of these biologically important channels.
voltage-gated chloride channels
THE BASIC STRUCTURE OF EVERY biological membrane is a
lipid bilayer that is virtually impermeable to charged
molecules. Ion transport through membranes is
achieved by specialized membrane proteins that permit crossing of ions by different mechanisms of decreasing the Born barrier and are usually classified as
channels, carriers, or pumps according to the transport
mechanism (47). Certain ion channels have been extensively studied over the last hundred years, and
considerable progress has been made in the last few
years to provide a detailed understanding of the molecular basis of function of these particular channels
(6, 11, 25, 30).
In contrast, the majority of transport proteins remain little studied, and for these the basic function is
still insufficiently understood. One of these neglected
protein classes is that of the voltage-gated anion or
chloride channels, anion-selective channels that display voltage-dependent transitions between open and
Address for reprint requests and other correspondence: Ch. Fahlke,
Institute of Physiology, RWTH Aachen, Pauwelsstr. 30, D-52057
Aachen, Germany (E-mail: [email protected]).
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closed states and do not depend on the binding of a
ligand to open (29) These chloride channels do not play
major roles in the initiation and spread of excitation in
excitable membranes and the neuronal synaptic transmission, but they regulate excitability in nerve and
muscle by adjusting length constants and input resistance (1, 29). Anion channels often stabilize the membrane potential of excitable cells by providing an additional conductance, reversing close to the resting
potential. They also fulfill important housekeeping
functions like volume regulation (46) and pH regulation in organelles (2) and are crucial for transepithelial
solute transport (28, 60).
Voltage-gated chloride channels are difficult to study
in native preparations. Molecular cloning and subsequent heterologous expression were major breakthroughs in the investigation of this class of ion channels, and this was first accomplished by Jentsch and
colleagues in 1990 (34), when they cloned a chloride
channel, ClC-0, from the electric organ of Torpedo
marmorata. As a large number of homologs were subsequently identified, ClC-0 became the ancestor and
the gene precursor of a novel channel family of voltagegated chloride channels, the ClC family. The ClC fam-
0363-6127/01 $5.00 Copyright © 2001 the American Physiological Society
http://www.ajprenal.org
INVITED REVIEW
ily presently represents the largest known gene family
coding for anion channels (32, 33). At least nine human
isoforms (ClC-1 to ClC-7, ClC-Ka, and ClC-Kb) are
expressed in various tissues. Some of them are known
to play important roles in the function of certain organs, whereas for others the physiological importance
has not yet been identified. ClC-1 is almost exclusively
expressed in adult skeletal muscle fibers and is responsible for its large chloride conductance at resting potential (57). Mutations in this isoform cause myotonia
congenita (24, 39), a muscle disease characterized by
stiffness on sudden forceful movement. ClC-2 is ubiquitously expressed and seems to fulfill various tasks in
several organs. ClC-2 was originally proposed to be
an ubiquitously expressed volume-sensitive chloride
channel (26), but this role has been disputed in the last
few years (38). In neuronal cells, heterologous expression of ClC-2 decreases internal chloride concentration,
suggesting an important role of this isoform in adjusting the chloride equilibrium potential in neurons (56).
Furthermore, a role in transepithelial transport in the
gastrointestinal tract was recently demonstrated (8,
28). In cardiac myocytes, ClC-2 forms an inwardly
rectifying chloride channel with a possible role in the
regulation of excitability (13). ClC-3 was also suggested to be a ubiquitous volume-activated chloride
channel (12), but the discussion of this suggestion has
not yet been settled (38). Moreover, there is uncertainty about the functional properties of this isoform,
as some groups have reported successful heterologous
expression and others have not. ClC-4 and ClC-5 share
high sequence identity and are functionally very similar (22). In humans, ClC-5 is kidney specific, whereas
ClC-4 is expressed in muscle, brain, and heart. Mutations in the gene coding for ClC-5, CLCN5, cause
Dent’s disease, an inherited renal disorder associated
with hypercalciuria, nephrolithiasis, and low-molecular-weight proteinuria (40). ClC-5 is thought to form an
anion channel in endosomes of proximal tubule cells
that is crucial for proper acidification (10, 27, 52). A
similar role for ClC-4 appears possible, but this has not
been demonstrated as yet. ClC-6 and ClC-7 are ubiquitously expressed, but nothing is known about their
functional or physiological properties (5). ClC-K1 and
ClC-K2 in rats, or ClC-Ka and ClC-Kb in humans,
represent kidney-specific isoforms that are involved in
fluid resorption within the nephron (42, 55, 60). Genetic alterations of CLCNKB are responsible for type
III Bartter’s syndrome, a salt-wasting renal tubular
disorder causing hypovolemia, hyponatremia, and hypotension (55, 60).
ClC channels are clearly of high physiological importance, and understanding basic properties of ClC channels at the molecular level will provide important information regarding many physiological questions.
Studying ClC channels in heterologous expression systems provides the opportunity to combine cellular electrophysiology and recombinant DNA technology to
gain molecular insights into how chloride channels
work. In the following pages I will review the basic
properties of ClC channels and recent progress in our
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understanding of how anion channels fulfill their primary task, the selective transport of anions through
cellular and subcellular membranes.
PHYSIOLOGICAL ROLE OF CHLORIDE CHANNELS
The physiological task of ClC channels is quite different from the role of cation channels. Various cations
of similar sizes exist in intra- and extracellular solutions, and channels that selectively permit the passage
of Na⫹ or K⫹, but not of another ion, are key for many
physiological processes. In contrast, there are only two
anions, Cl⫺ and HCO3⫺, at millimolar concentrations in
biological media, and perfect discrimination seems not
to be crucial, as these anions are similarly distributed
over the cell membrane. Similarly, many anion channels are permeable to both chloride and bicarbonate.
Moreover, perfect anion-to-cation selectivity of chloride
channels appears necessary only in certain tissues. For
example, in excitable cells, a small Na⫹ or Ca2⫹ permeability through anion channels would provide a
component Na⫹ or Ca2⫹ conductance at resting potential and greatly disturb the normal excitation processes
(16). In contrast, in epithelial cells such a leak cation
conductance would be much less harmful, as in those
cells there are many active cation transport mechanisms.
FUNCTIONAL PROPERTIES OF CLC-TYPE
CHLORIDE CHANNELS
The various ClC isoforms do not share a high degree
of sequence identity, and they display a wide variety of
gating and permeation properties. Table 1 gives an
overview of functional properties of mammalian ClC
isoforms, illustrating the functional divergence within
this gene family. Figure 1 demonstrates representative
current recordings from four ClC isoforms: ClC-0 in T.
marmorata electric organ (Fig. 1A), hClC-1, the human
muscle isoform (Fig. 1B), rat ClC-2 (Fig. 1C), and
human ClC-4 (Fig. 1D). The four isoforms were chosen
because there is general agreement about the functional properties of these ClC channels. For each isoform, currents were recorded by whole cell patch-clamp
recordings from transiently transfected tsA201 cells.
Cells were held at 0 mV between pulses, and voltage
steps between ⫺165 and ⫹75 mV in 40-mV intervals
are shown. Conduction and gating properties differ
among the isoforms. ClC-0 and ClC-1 are open at 0 mV,
and currents deactivate on membrane hyperpolarization. ClC-0 displays a linear instantaneous currentvoltage relationship; hClC-1 is pronouncedly inwardly
rectifying. ClC-2 is closed at positive potentials and
activates on membrane hyperpolarization; the currentvoltage curve of the open channel is linear. ClC-4
outwardly rectifies and activates on membrane depolarization.
Selectivity among different anions, albeit physiologically not important, is of special importance for this
review as selectivity has provided important insights
into the function of the pores of ClC channels. Many
ClC channels display a Cl⫺ ⬎ Br⫺ ⬎ I⫺ (Table 1)
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INVITED REVIEW
Table 1. Functional properties of several ClC isoforms
ClC Isoform
Permeability Sequence
Open-Channel I-V
Relationship
Gating
ClC-0
Cl⫺ ⬎ Br⫺ Ⰷ I⫺, SCN⫺
Linear
ClC-0
Cl⫺ ⬎ Br⫺ ⬎ NO3⫺ ⬎ I⫺
Linear
rClC-1
hClC-1
rClC-2
SCN⫺ ⬎ Cl⫺ ⬎ Br⫺ ⬎ NO3⫺ ⬎ I Ⰷ CH3SO3⫺ Inwardly rectifying
Cl⫺ ⬎ SCN⫺ ⬎ Br⫺ ⬎ NO3⫺ ⬎ I⫺
Inwardly rectifying
Cl⫺ ⬎ Br⫺ ⬎ I⫺
Linear
hClC-2
Cl⫺ ⬎ I⫺ ⬎ Glu⫺
Linear
hClC-2
Cl⫺ ⬎ I⫺ ⬎ Glu⫺
Linear
Activation on
hyperpolarization
rbClC-2
Cl⫺ ⬎ ⫽ Br⫺ ⬎ I⫺ Ⰷ F⫺
Linear
rClC-3
I⫺ ⬎ Cl⫺ ⬎ Br⫺
Outwardly rectifying
Activation on
hyperpolarization
Inactivation on depolarization
rClC-3
I ⬎ Cl ⬎ Br
Outwardly rectifying
Activation on depolarization
gpClC-3
I⫺ ⬎ Cl⫺
Outwardly rectifying
Inactivation on depolarization
hClC-4
hClC-4
rClC-5
NO ⬎ Cl ⬎ Br ⬎ I
I⫺ ⬎ Cl⫺ ⬎ F⫺
NO3⫺ ⬎ Cl⫺ ⬎ Br⫺ ⬎ HCO3⫺ ⬎ I⫺
Outwardly rectifying
Outwardly rectifying
Outwardly rectifying
Activation on depolarization
Activation on depolarization
Activation on depolarization
rClC-K1
Br⫺ ⬎ Cl⫺ ⬎ I⫺
Outwardly rectifying
Activation on depolarization
⫺
⫺
⫺
3
⫺
⫺
⫺
⫺
Fast gate activating on
depolarization, slow gate on
hyperpolarization
Fast gate activating on
depolarization, slow gate on
hyperpolarization
Activation on depolarization
Activation on depolarization
Activation on
hyperpolarization
Activation on
hyperpolarization
Origin of Tested Channels
(Ref. No.)
Native channels,
reconstituted in planar
lipid bilayers (45)
Expressed in Xenopus
oocytes (34)
Expressed in Sf9 (51)
Expressed in HEK293 (16)
Expressed in Xenopus
oocytes (59)
Endogenous channels in
human immortalized
airway cells (54)
Heterologously expressed
in human immortalized
airway cells (54)
Expressed in Xenopus
oocytes (23)
Expressed in Xenopus
oocytes (37)
Expressed in CHOK1
(36)
Expressed in NIH3t3
(12)
Expressed in CHO-1 (22)
Expressed in CHO-1 (35)
Expressed in Xenopus
oocytes (58)
Expressed in Xenopus
oocytes (61)
ClC, voltage-gated chloride channel; I-V, current-voltage; Glu, glutamate; r, rat; h, human; rb, rabbit; gp, guinea pig; CHO, Chinese
hamster ovary.
permeability sequence, in contrast to other anion-selective channels, such as glycine and GABA receptors
or volume-sensitive chloride channels (4, 46). Obviously, there are functional differences between the ion
pores of members of the ClC family and those of many
other anion-selective channels. There are two exceptions, ClC-3 (12) and hClC-4 at an external pH (pHo) of
4.5 (35). The results for ClC-3 are presently a matter of
controversy (12, 21, 36, 37), as there are conflicting
published data and several groups have failed to reproduce these results because they could not functionally
express ClC-3.
Interestingly, for ClC-type chloride channels, the
anion permeability sequence is affected by changing
the pHo. Native chloride channels in frog muscle exhibit an anion permeability sequence of Cl⫺ ⬎ Br⫺ ⬎
NO3⫺ ⬎ I⫺ at physiological pHo, but an inverted one
(Cl⫺ ⬍ Br⫺ ⬍ NO3⫺ ⬍ I⫺) at pHo of 5.0 (31). For cloned
ClC channels, a similar result was reported for hClC-4.
Friedrich et al. (22) observed a Cl⫺ ⬎ I⫺ permeability
sequence at physiological pH, whereas Kawasaki et al.
(35), using the same clone in a similar expression
system, observed an I⫺ ⬎ Cl⫺ sequence at pHo of 4.5.
These examples establish that anion selection by this
class of channels is under the control of a titratable
side chain.
Fig. 1. Representative current recordings from several voltage-gated
chloride channels (ClC) isoforms: ClC-0 (A), hClC-1 (B), rClC-2 (C), and
hClC-4 (D). Currents were recorded by using tight whole-cell recordings
from tsA201 cells, transiently transfected with coding regions of various
ClC isoforms subcloned into the pRc/CMV vector as previously described (16). Cells were bathed in a standard external solution containing (in mM) 140 NaCl, 4 KCl, 2 CaCl2, 1 MgCl2, and 5 HEPES, pH 7.4,
and perfused with a standard internal solution (in mM: 130 NaCl, 2
MgCl2, 5 EGTA, and 10 HEPES, pH 7.4). A holding potential of 0 mV
was chosen, and voltage steps between ⫺165 and ⫹75 mV were applied
as shown in the pulse diagram (inset).
INVITED REVIEW
FUNCTIONAL CHARACTERIZATION OF THE CLC ION
CONDUCTION PATHWAY
A number of electrophysiological studies using permeant and impermeant anions have provided insights
into the functions of the pore and the selectivity mechanism of ClC channels. These studies, pioneered by
Miller and White (45), focus on two closely related ClC
isoforms, ClC-0 and ClC-1. For other ClC channels,
such an analysis is still missing. Miller and White
probed native ClC-0 channels in planar lipid bilayers
with various anions. They investigated single-channel
and macroscopic current amplitudes at several concentrations of Cl⫺ and other anions, as well as reversal
potentials under bi-ionic conditions. In their hands,
ClC-0 is permeable only to Cl⫺ and Br⫺ and is blocked
by other anions, including I⫺ and SCN⫺. ClC-0 exhibits
a low-affinity binding site for Cl⫺ [dissociation constant (KD) ⫽ 75 mM], and several tests revealed that
the channel is only singly occupied (absence of anomolous mole fraction, concentration independence of reversal potentials under bi-ionic conditions, and concentration independence of the parameters describing the
blocking action of SCN⫺). A later study of ClC-0 channels heterologously expressed in Xenopus laevis oocytes (48) reported anomolous mole fraction behavior of
macroscopic currents and concluded that this ClC isoform exhibits multiple occupancy. The reason for these
different experimental results is unclear.
The muscle ClC isoform, ClC-1, is similar to ClC-0 in
many respects but differs in others. For ClC-1, the
reversal potential determined under bi-ionic conditions
with a constant concentration ratio changes with alterations of the absolute concentration (18). This result is
clear evidence that ClC-1 is multiply occupied. Various
permeant anions, such as I⫺, SCN⫺, NO3⫺, or Br⫺,
block Cl⫺ current through hClC-1 by binding to sites
within the ion conduction pathway with higher affinity
than does Cl⫺ (16, 18, 51). These anions remain at this
site longer, and this feature reduces their permeability
and accounts for the observed block of Cl⫺ current (15,
16, 31, 49). Cl⫺ current block is accompanied by kinetic
effects that depend on the side of the membrane to
which the blocking anions are applied. On membrane
hyperpolarization, muscle chloride channels deactivate
with a biexponential time course (20). Although the
time constants are almost voltage independent, the
relative amplitude of the fast-deactivating, slow-deactivating, and nondeactivating current components are
highly voltage dependent. External application of I⫺,
SCN⫺, NO3⫺, or Br⫺ changes the voltage dependence of
the fractional current amplitudes but leaves the time
constants of deactivation unaffected (18, 51). In contrast, internal application slows the deactivation time
constants (18, 51). These results demonstrate that
there are at least two distinct binding sites within the
hClC-1 ion conduction pathway (16). The two binding
sites differ in their relative anion selectivity; i.e., the
extracellular site exhibits an affinity sequence of
SCN⫺ ⬎ I⫺ ⬎ NO3⫺ ⬎ CH3SO3⫺ ⬎ Br⫺ ⬎ Cl⫺ ⬎ F⫺ and
the intracellular one of I⫺ ⬎ NO3⫺ ⬎ SCN⫺ ⬎ Cl⫺ ⬎ F⫺
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(18). By using the characteristic kinetic changes induced by external and internal I⫺, the KD of the internal and external binding sites could be independently
determined. The KD values so obtained are in the
millimolar range (18). Because iodide binds more
tightly than chloride, this value represents a lower
limit for the chloride KD, indicating that hClC-1 binds
anions with affinities that are more than an order of
magnitude lower than the permeant ion affinities of
potassium (62) and calcium channels (9). Another important result of this study is that the concentration
and voltage dependences of the effect of I⫺ on the
fast-deactivating component are distinct from those on
the slow-deactivating and the nondeactivating components. For all three current components, the KD (0 mV)
as well as the voltage dependence of the KD are different (18). These results suggest that hClC-1 undergoes
conformational changes of the ion conduction pathway
during transition between the three kinetic states.
To study the functional basis of the distinct relative
anion permeabilities of various anion channels, we
studied the interaction of anions within the ion conduction pathway for several ClC channel constructs
exhibiting distinct anion permeability sequences. The
first ClC channel construct demonstrated as exhibiting
a larger permeability for I⫺ than for Cl⫺ was a mutant
ClC-1 channel that carries a disease-causing G230E
mutation (16). G230E hClC-1 exhibits an inverted anion permeability sequence of SCN⫺ ⬎ NO3⫺ ⬎ I⫺ ⬎
Br⫺ ⬎ Cl⫺. Experiments revealed that neither external
nor internal I⫺ blocks Cl⫺ currents, although its ability
to change gating properties indicates that I⫺ still binds
better than Cl⫺ (16). The effect of I⫺ on various ClC
channel constructs that exhibit a larger I⫺ than Cl⫺
permeability is, however, not uniform. Figure 2 shows
representative voltage-clamp current recordings and
instantaneous current amplitudes in external Cl⫺ and
in external I⫺ for four ClC channel constructs: WT
hClC-1, G230A hClC-1, K231A hClC-1, and the ClC-3/
ClC-1 chimera described above. WT hClC-1 exhibits a
PI/PCl of 0.34, where P is permeability, while all other
constructs exhibit a higher I⫺ than Cl⫺ permeability
(PI/PCl ⬃2.5) (21).WT hClC-1 displays a I⫺ block over
the whole voltage range, accompanied by a shift of the
current reversal potential to the right, causing the
Cl⫺ ⬎ I⫺ permeability sequence of this ClC isoform.
For G230A hClC-1, there is a similar blocking action,
and the I⫺ conductivity at positive potentials is very
small, but the reversal potential shifts in the opposite
direction. For cells expressing K231C hClC-1, Cl⫺ currents in the negative-voltage range are blocked by I⫺,
but the I⫺ current at positive potentials is larger than
the Cl⫺ current. The fourth illustrated construct is a
rClC-3/hClC-1 chimera that was generated by replacing the D3-D5 segment of hClC-1 with that of rClC-3
(21). Over the whole voltage range, no blocking action
of I⫺ was observed (15). Moreover, the changes in
current amplitudes were similar to the predictions of
the Goldman-Hodgkin-Katz equation (29), suggesting
that different anion fluxes only minimally interfere
with each other.
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INVITED REVIEW
higher anion flux at positive potentials. At negative
potentials, the presence of I⫺ markedly reduces channel conductance. This indicates a clear interaction between simultaneously bound anions. For the chimera,
the virtual absence of interaction between the two
anions suggests that a single site determines ion conduction through the pore.
Other anions that can be also used to probe the ionic
pore of ClC channels are F⫺ and CH3SO3⫺. Although
ClC-0 and ClC-1 are not measurably permeant for F⫺
or CH3SO3⫺ (15, 51), ClC-4 exhibits a permeability for
these two anions (35). These results allow predictions
about the pore narrowing of different isoforms.
A MODEL FOR THE MECHANISM OF SELECTIVITY
IN CLC-TYPE CHLORIDE CHANNELS
Fig. 2. Effect of changing the external anion composition for 4
different ClC constructs: WT hClC-1 (A and B), G230A hClC-1 (C and
D), K231C hClC-1 (E and F), and a rClC-3/hClC-1 chimera (D3–D5;
G and H) (21). A, C, E, and G: whole cell recordings from the different
channels transiently transfected in tsA201 cells. B, D, F, and H:
voltage dependence of the instantaneous current amplitudes in standard external solution (F) and from the same cell after the extracellular solution has been changed to (in mM) 140 NaCl, 4 KCl, 2 CaCl2,
1 MgCl2, and 5 HEPES, pH 7.4 (䊐).
The above-mentioned four examples illustrate the
different pattern of anion binding within the ion conduction pathway of channels with similar relative I⫺
permeabilities. All these observations can be explained
in the context of more than one functionally distinct
anion binding site within the pore. The result that an
external I⫺ block of G230A hClC-1 is similar to that of
WT implies that the external binding site is almost
unaffected by the mutation. In contrast, the I⫺ affinity
of the internal site is reduced, resulting in diminished
block by intracellularly applied I⫺ (Fahlke, unpublished observations). The observed leftward shift of the
reversal potential on substitution of Cl⫺ by I⫺ is due to
this alteration of the internal binding site. I⫺ is less
sticky and is able to translocate to the internal medium
more easily than in WT channels, causing the I⫺ ⬎ Cl⫺
permeability sequence of the mutant. In K231C
hClC-1, obviously both binding sites are affected. The
I⫺ conductivity at positive potentials is higher than for
Cl⫺, implying that the external binding site is displaying a reduced absolute anion affinity and permitting a
Selection between anions and cations can be easily
explained with electrostatic interactions between ions
and the channel protein. Cation channels use negative
charges in pore vestibules to attract cations and to
repell anions, and it is easy to imagine that anion
channels use a similar mechanism, a positive electrostatic potential to prevent the permeation of cations
and thus be anion selective. Studying the mechanism
by which ClC channels select among anions has provided much more information about basic pore properties than the anion-cation selection process.
Ions in aqueous solution are surrounded by a shell of
water molecules. The electrostatic interaction of the
water dipole with the ion charge is quite strong, with
free energy values in the range of 100 kcal/mol. For the
majority of selective ion channels, the first step in ion
permeation is a partial dehydration, reducing the size
of the permeating complex and therefore allowing the
ion without water molecules, or with a few, to pass a
confined pore narrowing (Fig. 3A). The energy necessary for dehydration is provided by the binding of
dehydrated ions to the amino acids of the ion conduction pathway. For hClC-1, F⫺, the smallest tested
anion, is almost impermeant whereas Cl⫺, the anion
with the next larger diameter, has the highest permeability among all ions tested. This behavior indicates
that a dehydration step is occurring during anion permeation through this channel and is critically involved
in selection among anions. Binding of anions within
ClC channel pores occurs with low affinity, and it thus
appears reasonable that the interaction of the channel
pore with the permeating anion is dominated by electrostatic forces. These features demonstrate the feasibility of using the Eisenman approach to describe anion binding within ClC channel pores. The Eisenman
approach (14, 29) to describing the binding of ions
within pores takes only electrostatic interactions and
dehydration energies into account, and this simplification allows an easy mathematical treatment of channel
ion binding. This theory predicts that, for a given set of
ions, only a certain number of permeability sequences
exist, the so-called “Eisenman sequences.” Within
these sequences, two extreme cases can be distinguished. One is that the binding site provides a weak
INVITED REVIEW
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Fig. 3. A: graphical illustration of the postulated passage of anions through a ClC channel pore. The hydrated
anion dehydrates by binding to a side within the pore, then passes the pore narrowing, binds to a distinct side, and
leaves the pore after rehydration. B: primary sequence regions that were identified to determine anion permeation
and selectivity in ClC-type chloride channels (17, 21). Transmembrane domains D1/D2 and D6–D12 are shown as
blank rectangles, and only the region between D3 and D5 is shown in more detail. Residues, the exchange of which
alters pore properties, are shown in white, black, and gray circles. Residues in gray circles denote those accessible
to hydrophilic methanethiosulfonate (MTS) reagents from the external solution; those in dark circles are accessible
from the internal solution. Residues in open circles represent positions at which substituted cysteines cannot be
functionally modified by MTS residues. With D5, nonaccessible residues are not shown. The 3 likely pore-forming
regions (P1, P2, and P3) are shown as rectangles with dotted borders. N, NH2 terminus; C, COOH terminus.
electrostatic field. In this case, the dehydration energy
is the dominating factor of binding. A weak binding site
will therefore prefer large anions over small ones as
the dehydration energy of these anions is smaller. The
other case is an electrostatically strong binding site, in
which the electrostatic term will dominate. Such a site
will prefer small anions over larger ones because they
provide smaller electrostatic energy terms. For hClC-1,
larger and polyatomic anions bind with higher affinities to sites within the ionic pore. Therefore, according
to the Eisenman theory, binding sites within the
hClC-1 pore are weak-field-strength sites. They provide little electrostatic binding energy, and binding
selectivity is largely determined by the dehydration
energy; thus they prefer larger anions over smaller.
The better binding of polyatomic anions such as SCN⫺,
NO3⫺, and CH3SO3⫺ suggests that hydrophobic components contribute to the binding sites. HClC-1 selects
among SCN⫺, I⫺, NO3⫺, Br⫺, and Cl⫺ by selective
binding to a low-affinity binding site. The tighter binding of certain anions reduces their turnover, and this
feature decreases the permeability as well as the conductivity of these anions (15, 31). Such a selectivity
mechanism is not possible for singly occupied pores, as
shown by Bezanilla and Armstrong (3), but is feasible
for ClC-1 because it is multiply occupied. The proposed
selectivity process of hClC-1 has the interesting feature of producing a selectivity sequence that prefers
small anions over large ones (and would be called a
strong-field interacting site in Eisenman terminology)
by means of selective binding to binding sites that are
weak-field interacting sites, i.e., that prefer large and
polyatomic anions over small anions. The weak interacting sites within the hClC-1 pore are unable to provide the energy necessary to dehydrate F⫺, and the
hydrated F⫺ cannot permeate the narrow part of the
pore as it is too bulky. In the context of this reasoning,
the increased F⫺ permeability of ClC-4 (35) may be
explained by suggesting a larger pore narrowing. If
hClC-4 exhibits a larger diameter of the pore narrowing than ClC-1, hydrated F⫺ could permeate hClC-4
but not hClC-1. Experiments with G230E hClC-1 demonstrated that channels with a I⫺ ⬎ Cl⫺ permeability
sequence can also exhibit binding sites preferring large
and polyatomic anions (16). A changed permeability
selectivity is therefore not due to changed binding
affinities. The major difference between ClC channels
with Cl⫺ ⬎ I⫺ and those with I⫺ ⬎ Cl⫺ is that the latter
do not exhibit a block by permeant anions. The following selectivity mechanism is in agreement with all
published experimental results.
ClC-type chloride channels select among ions by selective binding to several anion-selective binding sites.
All these sites prefer large or polyatomic anions over
small uniatomic ones; they are weak interacting sites
in Eisenman terminology (14, 29). By binding to these
anionic sites, anions dehydrate and the smaller radius
of dehydrated anions permits passage through a narrow pore constriction. Some anions simply do not permeate as they are too large to pass; for all others, the
binding to and unbinding from these sites is crucial in
determining passage of the anion through the channel.
In channels with Cl⫺ ⬎ I⫺ or Cl⫺ ⬎ NO3⫺ selectivity,
the rate-limiting step in anion permeation is the dissociation of anions from one of these sites. Anions that
bind with higher affinity to this site are less permeant
and cause block of current flow by anions that bind
with lower affinity. In channels with I⫺ ⬎ Cl⫺, or NO3⫺
Cl⫺ selectivity, the association, not the dissociation, of
permeant anions to one of these binding sites is the
rate-limiting step. For this reason, the permeability
sequence in these channels follows the affinity sequence of the binding sites. ClC channels with a I⫺ ⬎
Cl⫺ permeability sequence differ from those with
Cl⫺ ⬎ I⫺ in the absolute value of the interaction energy
between binding site and anion, not in the selectivity of
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INVITED REVIEW
these binding sites. As shown in Fig. 2, a single binding
site with such properties is sufficient to endow an
anion channel with larger permeability for I⫺ than for
Cl⫺. This again demonstrates the variability of structural changes that can cause an inversion of the permeability sequence of ClC-type chloride channels.
The selectivity mechanism of anion channels is
clearly distinct from that of voltage-gated K⫹ and Ca2⫹
channels. Compared with the elegance with which
these channels perform their extremely challenging
task, selection in ClC-type chloride channels appears
almost awkward. The reason for such an “imperfection” is clearly that selection among different anions is
biologically not important. What could be the physiological importance of anion channels with a Cl⫺ ⬎
Br⫺ ⬎ I⫺ selectivity sequence compared with others
exhibiting the inverse sequence? We do not know as
yet, but a possible answer emerges from the abovementioned selectivity mechanism. As discussed above,
the two types of anion channels differ in the absolute
value of anion affinity of their ion conduction pathway.
Thus it appears possible that channels with Cl⫺ ⬎
Br⫺ ⬎ I⫺ sequence will be more selective for anions
over cations because of the higher electrostatic potentials of their ion conduction pathway. The functional
properties of ClC-1 responsible for the large Cl⫺ conductance in skeletal muscle are in agreement with this
hypothesis. ClC-1 has a Cl⫺ ⬎ I⫺ permeability sequence and exhibits a very small relative cation
permeability, as even a small cation conductance at
resting potential would drastically alter muscle excitability. Clearly, evaluation of this hypothesis awaits
accurate determination of the relative cation permeabilities of various ClC isoforms.
IDENTIFICATION OF MOLECULAR DETERMINANTS
OF ION SELECTIVITY IN CLC CHANNELS
Functional studies of the anion selection process of
ClC channels provide indirect information about the
structural elements involved. We know that ClC channels exhibit weak Eisenman interaction sites that interact with permeating anions with low affinity. The
obvious dependence of the anion permeability sequence
on pHo demonstrates that titratable sites are critically
involved in anion binding. As all known ClC channels
are anion selective, we expect an evolutionarily conserved anion-selective pore that exhibits positively
charged side chains protruding into the aqueous ion
conduction pathway to create a positive electrostatic
potential. ClC channels distinguish among anions by
selective binding to low-affinity binding sites, and
therefore we expect that the anions do not make intimate contact with structures within the pore wall.
Allosteric and other long-range effects of point mutations can have considerable influence on the interaction between the channel and permeant anions and
thus on the selectivity and single-channel current amplitude. These properties pose a particular problem for
approaches using a combination of site-directed mutagenesis and cellular electrophysiology to identify
pore residues. The effect of a single amino acid exchange on ion conduction properties (16, 41, 48, 64)
does not define this region as contributing to the formation of the ion conduction pathway, and special care
has to be taken in defining pore-forming regions using
such an approach.
There are nevertheless certain regions within the
ClC primary sequence that fulfill all the criteria for a
selectivity filter and pore. A disease-causing mutation,
found in a family with Thomsen’s dominant myotonia
(24), resulting in the substitution of a glycine by glutamic acid COOH terminal to the third transmembrane domain (G230E), dramatically alters the ion
conduction pattern of hClC-1 and inverts the anion
permeability sequence form Cl⫺ ⬎Br⫺ ⬎ I⫺ to I⫺ ⬎
Br⫺ ⬎ Cl⫺ (16). In the framework of the proposed
selectivity mechanism described above, the result suggests that amino acid 230 is close to ion binding sites
within the pore. An additional negative charge at this
position would weaken the interaction with the permeant anion and increase the off-rate constant. This
could explain the higher permeability of larger and
polyatomic anions. It would make the electrostatic
potential within the pore less positive, thus accounting
for the observed increase in cation permeability. G230
is part of a highly conserved eight-amino acid stretch
present in every ClC channel identified (GKxGPxxH)
(21) (Fig. 3B), and such an evolutionary conservation is
expected for a region that represents a core structural
element of the pore of ClC channels. We tested this
hypothesis by a combination of cellular electrophysiology, site-directed mutagenesis, and chemical modification. Every amino acid substitution made within this
region affects the anion selectivity of hClC-1 (21). In
two of the eight positions (231 and 237), there is a clear
dependence between substituted charge and anion selectivity (21). Such an association implies a close interaction between the side chain and the permeating
anion and suggests that these two amino acid side
chains, K231 and H237, protrude into the ionic binding
sites (Fig. 3B). Of eight single substituted cysteines in
the P1 region, three (K231C, P234C, and H237C) react
with methanethiosulfonate (MTS)-reagents MTSethyltrimethylammonium (MTSET) and MTS-ethylsulfonate (MTSES), resulting in channel block (21).
K231C is only reactive to extracellular MTS reagents,
whereas P234C and H237C only react with intracellular reagents. As a short stretch of three amino acids
cannot span the entire lipid bilayer, this result is only
possible if all three cysteines protrude into an aqueous
pore that connects the extra- and intracellular medium.
MTS ethylammonium (MTSEA), which has the same
charge as MTSET, did not result in channel block but
in a change of channel gating and selectivity. Both
MTSET (positively charged) and MTSES (negatively
charged) have a diameter of 6Å (which is larger than
that of MTSEA and the estimated minimum pore diameter of hClC-1), and they effectively block current
flow. These results imply that the P1 region lines the
narrowest part of the ClC pore.
INVITED REVIEW
The P1 region is responsible for the high anion-tocation selectivity of hClC-1 (21). Several mutations in
the first four residues of the P1 region (GKEG in
hClC-1) change the cation permeability. Estimating
the electrostatic potentials near substituted cysteines
by comparing the reaction rate with MTSES and
MTSET (7) reveals that the cavity formed by the P1
region is inherently anion selective. Because two of
these cysteine substitutions (K231C and H237C) remove a positive charge, the electrostatic potential in
the WT hClC-1 will be even more positive and could be
fully responsible for the low cation permeability of ClC
channels (21). The GKEG motif within the P1 region
appears to be of special importance for ion selectivity in
ClC channels. Several results indicate that K231
projects into the ionic pore, and similar experimental
approaches suggest that the negatively charged side
chain of E232 points away from the ion conduction
pathway. Because of the flanking glycine residues, the
position of this two-amino acid dipole is flexible and
may be affected by interaction with other parts of the
ClC sequence. Small positional changes because of
alteration of the conformation of interacting protein
parts may alter the electrostatic potentials close to the
P1 region and account for the observed isoform-specific
permeation differences in ClC channels. The two positively charged side chains (K231 and H237) that
project into the pore represent likely anion-binding
sites. The combination of cationic groups and hydrophobic side-chain and backbone structures in the putative narrow pore can account for the experimentally
observed higher binding affinity for large and polyatomic anions (16, 18). Although the P1 region exhibits
accessible charged amino acid side chains, it cannot
account for the effect of external acidifcation on anion
permeability, as the exposed basic amino acids will not
lose their charge at acidic pH. Moreover, it is obvious
that this short stretch of amino acids cannot form an
ion conduction pathway by itself. Searching for additional pore-forming regions, we focused on highly conserved sequence motifs. Within D5, we have identified
another highly conserved amino acid stretch, GVLFSI
in ClC-1 (designated as the P2 region) (Fig. 3B). Exchange of single amino acids in P2 affects the anion
permeability sequence. Substituted cysteines at certain positions within this motif are accessible to hydrophilic MTS reagents, and that within P2, there is a
change of sidedness of MTS modification; i.e., at the
NH2-terminal end, substituted cysteines are accessible
from the internal solution, and at the COOH-terminal
end, from the external solution. We concluded from
these results that this region, together with the P1
region, lines the narrow part of the pore (21) Using
similar approaches, we showed that a highly conserved
eight-amino acid motif (P3) located in the linker between transmembrane domains D2 and D3 contributes
to the formation of a pore vestibule facing the cell
interior (17) (Fig. 3B). The role of these motifs has also
been demonstrated for other isoforms (22, 63), supporting the notion that they represent evolutionarily conserved pore regions.
F755
To gain insights into the dimensions of the pore, we
used cysteine-specific reagents of different sizes (17).
The reactivity of MTS-propyltriethylammonium (MTSPTrEA) and monobromotrimethyl-ammoniobimane
(qBBr) with the substituted cysteines at these positions reveals that the pore diameter is wider than 10 Å
at the level of K231 (within the P1 region) and G190C
(within the P3 region) and that E193 (P3 region) and
H237 (P1 region) are located in pore regions that are
more restricted. These marked differences in the diameter of pore-forming regions imply that ClC channels
exhibit a pore architecture quite similar to that of
certain cation channels: a narrow constriction containing major structural determinants of ion selectivity is
neighbored by wide vestibules on both sides of the
membrane. The result that Cys231 can be modified with
large cysteine-specifc reagents is astonishing as cysteines at position 231 of both subunits of a functional
dimeric hClC-1 channel can form a disulfide bridge
(19). These two experimental results suggest that the
P1 region is very flexible. Another experimental finding that indicates a flexibile pore-forming region is the
absence of periodicity in the reactivity of substituted
cysteines within P3. Cysteines introduced between positions 189 and 193 are all reactive, and this could be
explained by conformational changes of this region
(17). Flexibile pore regions could explain the experimental results suggesting conformational changes of
the hClC-1 ion conduction pathway (18) and could be a
structural feature explaining the tight coupling of permeation and gating observed for ClC-0 (48) and ClC-1
(51).
Most probably, there are additional pore regions that
remain to be discovered. It is likely that certain regions
will remain elusive in mutagenesis experiments as
they do not interact with the permeant anions and thus
do not contribute to the selectivity process, and for
these novel approaches will be necessary.
PORE STOICHIOMETRY
ClC-0 exhibits unique single-channel behavior, with
two equally spaced and independently gated subconductance states. To account for this finding, Miller (44)
postulated that an individual channel exhibits two ion
conduction pathways, i.e., a novel double-barreled pore
stoichiometry for this channel. In the last few years,
several experimental results have been reported that
support the idea that a ClC channel consists of two
subunits and that each subunit forms an individual ion
pore (41, 43, 53). Nevertheless, there are some experimental results that appear to be incompatible with this
concept. For hClC-1, three independent experimental
lines of evidence support the idea that a major determinant of pore properties, the P1 region functionally
interacts with the corresponding region of the other
subunit (19) in a way that would be expected if the two
P1 regions form a single pore vestibule. This finding
was originally interpreted as evidence for a unipore ion
conduction pathway in ClC channels (19). One possible
explanation that could account for all presently known
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INVITED REVIEW
experimental results would be two protopores that
share a common vestibule, with important structural
determinants of ion permeation; another is that the P1
regions simply form adjacent walls of the two protopores. Recently, Ramjeesingh and colleagues (50) reported an exciting new finding that could provide a
third possibility for resolving this controversy. They
showed that ClC-2 channels purified from Sf9 cells
form functional channels in a dimeric as well as in a
tetrameric subunit stoichiometry. Reconstituted in
planar lipid bilayers, tetrameric channels exhibited
two conductance levels, and dimeric only a single one.
SUMMARY AND OUTLOOK
Work in the last several years has provided the first
insights into the function and structural determinants
of the ion pore of ClC-type chloride channels. Our
understanding of this family of channels is clearly just
beginning. Additional experiments, employing functional, molecular, and structural approaches, will be
necessary to fully understand the molecular basis of
anion permeation. Because of the widespread importance of this channel family in normal and pathological
physiology, these results will have a major impact on
biology and medicine.
I thank Dr. J. P. Johnson for helpful discussions and critical
reading of the manuscript and Dr. Al George for continuous support.
I am very grateful to Dr. T. J. Jentsch (Hamburg) and Dr. J. E.
Melvin for kindly providing me with ClC-0 and rClC-2.
This work was supported by the Deutsche Forschungsgemeinschaft (Heisenbergstipendium Fa 301/3–1, Fa 301/4–1) and the
Muscular Dystrophy Association.
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