Physiol Rev 88: 351–387, 2008;
doi:10.1152/physrev.00058.2006.
CLC-0 and CFTR: Chloride Channels Evolved
From Transporters
TSUNG-YU CHEN AND TZYH-CHANG HWANG
Center for Neuroscience and Department of Neurology, University of California, Davis, California;
and Department of Medical Pharmacology and Physiology, Dalton Cardiovascular Research Center,
University of Missouri, Columbia, Missouri
V.
VI.
VII.
VIII.
IX.
Introduction
General Mechanisms of Ion Channels and Transporters
CLC Transport Protein Family
Bacterial CLC Protein: A Chloride-Proton Antiporter
A. Structure of CLC-ec1
B. Mechanistic function of CLC-ec1
CLC-0: A Prototype Channel in the CLC Family
A. Introduction
B. Channel conductance and ion permeation of CLC-0
C. Gating properties of CLC-0
D. Mechanistic models for CLC-0 fast-gating
E. Unsettled issues and future directions
ABC Transporter Family
Structure/Function of ABC Transporters
A. Structural characteristics of the nucleotide binding domain in ABC transporters
B. NBDs form dimeric structures
C. Crystal structures and working model of ABC proteins
CFTR Chloride Channel
A. Introduction
B. Crystal structures of CFTR’s NBD1
C. CFTR function
D. Roles of ATP hydrolysis in CFTR gating
E. Coupling of NBD dimerization to gating transitions
F. Are the two ATP-binding sites functionally distinct?
G. Outstanding issues and future directions
Evolution and Speciation
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II.
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IV.
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Chen T-Y, Hwang T-C. CLC-0 and CFTR: Chloride Channels Evolved From Transporters. Physiol Rev 88: 351–387,
2008; doi:10.1152/physrev.00058.2006.—CLC-0 and cystic fibrosis transmembrane conductance regulator (CFTR) Cl⫺
channels play important roles in Cl⫺ transport across cell membranes. These two proteins belong to, respectively,
the CLC and ABC transport protein families whose members encompass both ion channels and transporters.
Defective function of members in these two protein families causes various hereditary human diseases. Ion channels
and transporters were traditionally viewed as distinct entities in membrane transport physiology, but recent
discoveries have blurred the line between these two classes of membrane transport proteins. CLC-0 and CFTR can
be considered operationally as ligand-gated channels, though binding of the activating ligands appears to be coupled
to an irreversible gating cycle driven by an input of free energy. High-resolution crystallographic structures of
bacterial CLC proteins and ABC transporters have led us to a better understanding of the gating properties for CLC
and CFTR Cl⫺ channels. Furthermore, the joined force between structural and functional studies of these two
protein families has offered a unique opportunity to peek into the evolutionary link between ion channels and
transporters. A promising byproduct of this exercise is a deeper mechanistic insight into how different transport
proteins work at a fundamental level.
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I. INTRODUCTION
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II. GENERAL MECHANISMS OF ION CHANNELS
AND TRANSPORTERS
Membrane transport mechanisms have been classified thermodynamically based on their ability to mediate
“passive” or “active” transports (126, 187, 303) (Fig. 1A).
Passive transport moves a solute across the membrane
from a side of high electrochemical potential to the other
side that is of low electrochemical potential. Two types of
membrane proteins, facilitated transporters and ion channels, transport substrates across the membrane “passively.” However, the fundamental mechanism by which
these two passive transport proteins work is thought to be
very different. Binding of the substrate to a facilitated
transporter from one side of the membrane induces conformational changes that expose the substrate to the
other side of the membrane. The concentration gradient
of the substrate thus drives the cycle of the conformational changes that is coupled to the movement of the
substrate. Because of this coupling of substrate movement to a cycle of protein conformational changes, the
transport rate for a facilitated transporter is relatively
low. On the other hand, ion channels contain an “aqueous” pore that allows diffusions of substrates (in this
case, permeant ions) to occur (Fig. 1A, left). Once the
pore is opened (by a process called “gating”), ions diffuse
through the opened pore at a very high rate, normally on
the order of 106/s or higher. Nevertheless, either passive
process dissipates the electrochemical gradient across
the membrane built up by the action of an active transporter that converts one form of energy (e.g., the chemical potential in ATP) to another (e.g., the electrochemical
gradient across cell membranes). Contrary to passive
transport mechanisms, an active transporter molecule
can pump ions across the membrane against the electrochemical gradient. To do so, an input of free energy is
required. For some transport molecules, such as Na⫹-K⫹ATPase or members of the ABC transporter family of
which CFTR is a member, ATP is hydrolyzed during the
transport cycle, and the energy directly harvested from
ATP hydrolysis is used to do the work. This type of
transport mechanism is called primary active transport
because the energy comes directly from ATP, which is
considered the energy currency in cells (Fig. 1B). Another
class of transport proteins mediates the net transfer of
one solute against its electrochemical gradient by using
the energy derived from the electrochemical gradient of
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Ion channels and transporters are integral membrane
proteins that transport ions and small molecules across
cell membranes. In doing so, they control many critical
physiological processes, including determining the voltage across cell membranes, maintaining the cell volume,
controlling the release of hormones and neurotransmitters, and regulating the secretion or absorption of important ions and substances, to name a few. Traditionally, the
transport processes of these two classes of molecules
were viewed from very different angles. Ion channels
were thought to contain a water-filled pore through which
ions diffuse down their electrochemical gradient. In contrast, molecules or ions moved by transporters were
thought to first bind to the transporter molecules, with the
substrate binding then inducing conformational changes
that switch the binding site from one side of the membrane to the other. Many transport proteins “pump” substrates across cell membranes by coupling the substrate
translocation to an energy releasing process (187, 303). It
is generally believed that a large conformational change
of the transporter molecule occurs during the transport
process.
With the progress in the structural and functional
studies of membrane transport in the last 10 years or so,
the separation line between ion channels and transporters
has become more and more murky. It was discovered in
the mid 1990s that glutamate transporters can simultaneously work as chloride channels (83, 91, 267, 295, 325).
Recent studies showed that some toxin molecules,
when bound to a classical transporter molecule, Na⫹K⫹-ATPase, can convert this transporter into an ATPgated ion channel (19, 20). Molecular cloning of the ion
channels and transporters further raises the questions
regarding the distinction between ion channels and transporters. When the cystic fibrosis transmembrane regulator (CFTR), whose defect is responsible for the hereditary
disease cystic fibrosis, was cloned, it was recognized that
this protein belongs to a widely distributed ABC transporter family. However, functional studies revealed that
CFTR is a genuine ion channel (reviewed in Refs. 96, 290).
Another gene family, the CLC Cl⫺ channel/transporter
family (142, 146, 197), was recently shown to include Cl⫺
channels and Cl⫺-H⫹ antiporters (51, 76, 144, 211, 241,
246). In the CLC family, CLC-0, CLC-1, and CLC-2 from the
vertebrate species function as Cl⫺ channels, while their
bacterial homologs are transporters. Interestingly, other
mammalian members of this family, such as CLC-4 and
CLC-5, may also function as transporters. In contrast, in
the ABC transporter family, CFTR is the only member that
serves as an ion channel. It appears that nature has found
a way to convert transporter proteins into ion channels
through evolution. The goal of this article is to review
recent work that addresses the mechanistic operations of
CLC and CFTR Cl⫺ channels and their transporter partners from the same family. While delving into the evolutionary connection between ion channels and transporters, we hope to gain mechanistic insights into how these
proteins carry out the essential function of membrane
transport.
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CLC-0 AND CFTR
another solute. Thus solute A is transported against its
electrochemical gradient at the expense of dissipating the
electrochemical gradient of solute B. Two situations can
occur. If solutes A and B are transported in the same direction, then the transport mechanism is called “cotransport,”
or “symport.” If solutes A and B are transported in opposite
directions, then the transport mechanism is called “counter-
FIG. 1. A: ion channels versus transporters. The cartoon depicts the
difference in the traditional concepts of ion channels and transporters.
For ion channels (left), ions diffuse through the open pore, which is
thought to be controlled by one gate. For transporters (right), because
an open pore is not allowed at any time, the transport pathway must be
guarded by at least two gates. The opening of the two gates is coordinated so that no open pore is allowed. The “1” represents the transported substrate that binds to the binding site in the transport pathway.
B: primary active transport mechanism. Energy directly harvested from
the hydrolysis of ATP (the energy currency in cells) is used to pump the
substrate across the membrane against an electrochemical gradient.
C: secondary active antiport mechanism. The two substrates, “1” and “2”,
are transported in opposite directions. The direction of the transport
cycle (“1” out and “2” in, or “1” in and “2” out) depends on the electrochemical gradients of these two substrates.
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transport,” or “antiport” (104) (Fig. 1C). The bacterial CLC
protein, such as the one from Escherichia coli (CLC-ec1),
is a Cl⫺-H⫹ antiporter (3).
Although passive and active transports are defined
thermodynamically, such definitions have an implication
for the structure of the substrate-transport pathway. For
ion channels, the ion diffusion through a water-filled pore
is required. For transporters, however, a water-filled,
open pore as seen in the open state of an ion channel is
not allowed because in such a situation substrate will
move down the electrochemical gradient. In the past several decades, physiologists envisioned that the ion-conducting pathway of ion channels should be controlled by
only one physical gate (Fig. 1A, left), while the substratetransport pathway of any transporter must be guarded by
at least two gates (Fig. 1A, right). Thus the opening of
either gate at any moment will not create an open pore in
transporters for substrate diffusion (104).
In this review, primary active transport and the antiport mechanism of secondary active transport will be the
focus of discussions because homologs of CFTR and CLC
Cl⫺ channels belong to these two categories of transport
mechanisms, respectively. Schematic transport cycles for
these two types of transport mechanisms are shown in
Figure 1, B and C. For the primary active transport (Fig.
1B), ATP first binds to the transport molecule, which also
serves as an enzyme to hydrolyze ATP. Hydrolysis of the
bound ATP and subsequent release of the hydrolytic products, ADP and Pi, complete the hydrolysis cycle that is
coupled to the movement of the substrate across the
membrane. In the antiporter mechanism shown in Figure
1C, the two transported molecules, “1” and “2”, are moved
in opposite directions; when solute 1 binds to the transporter on one side of the membrane, the other solute
(solute 2) must be released, and vice versa. The alternate
binding of the two substrates is important to form a
complete transport cycle. Investigators have thus speculated an outwardly facing and an inwardly facing structure for transporters as the two extreme states of a “rocking banana”-like motion shown in Figure 1C. One important feature in the antiport mechanism is the exchange
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III. CLC TRANSPORT PROTEIN FAMILY
The CLC family encompasses members widely distributed in various species ranging from bacteria to humans. These protein molecules play a variety of biological
roles, including maintenance of membrane potential, regulation of transepithelial Cl⫺ transport, and control of
intravesicular pH (142, 145, 146, 197, 326). The importance of the CLC family members is best illustrated by
numerous hereditary diseases caused by their defects,
such as myotonia congenita (101, 143, 156, 163, 351, 352),
Dent’s disease (100, 183, 184), Bartter syndrome (123, 160,
309), osteopetrosis (161), and idiopathic epilepsy (118),
and also from several gene knock-out studies in mice (36,
161, 235, 300, 328). The first member of the CLC gene
family, CLC-0, was identified and cloned from the electric
organ of the Torpedo ray (147, 227). Homology cloning
efforts in the 1990s identified nine members in mammalian species (CLC-1 to CLC-7, CLC-Ka and CLC-Kb), constituting three subfamilies (142, 197). CLC-0 and its mammalian homologs, CLC-1, CLC-2, CLC-Ka, and CLC-Kb
form one subfamily; they share ⬃50 – 60% amino acid
Physiol Rev • VOL
identity in the primary sequence. These members are Cl⫺
channels, and they are all present on the cell membrane to
control Cl⫺ flux and membrane potential. The second
branch of the family includes CLC-3, CLC-4, and CLC-5.
These proteins are located on the membrane of intracellular vesicles and are thought to be important in maintaining the pH of these vesicles (114, 217, 300), consistent
with their functional properties as Cl⫺-H⫹ antiporters.
However, recent work argued that CLC-3 is a doublebarreled Cl⫺ channel, residing on the postsynaptic plasma
membrane of certain neurons (330). That CLC-3 is a Cl⫺
channel on the cell membrane was also suggested by the
functional role of CLC-3 in regulating cell volume (74, 75,
343), although this issue has been controversial (200,
335). The third branch of the family contains CLC-6 and
CLC-7, which are expressed in various tissues. These two
proteins cannot be functionally expressed in heterologous expression systems (37). It has been reported that
they may be present also in the intracellular organelles to
regulate the vesicular pH (161).
Besides the CLC members cloned from mammalian
species, several members in bacteria (140, 198, 239), yeast
(99, 124, 282) and Caenorhabditis elegans (31, 222, 279,
301) have also been identified by searching the genomic
databases. The discovery of bacterial CLC members is
particularly important as the large quantity of bacterial
CLC proteins allow a direct structural approach. Although
the amino acid sequence identity between the bacterial
CLCs and those of the mammalian CLCs is only ⬃20%,
studies of mammalian CLC channels have shown that they
should adopt an overall similar structural architecture
(81, 86, 175, 252). Thus high-resolution crystal structures
from bacterial CLCs provide a useful framework to understand functional data of both bacterial and mammalian
CLCs.
IV. BACTERIAL CLC PROTEIN:
A CHLORIDE-PROTON ANTIPORTER
A. Structure of CLC-ec1
The E. coli CLC molecule (CLC-ec1) consists of ⬃400
amino acids (77), which correspond to the NH2-terminal
half of the vertebrate CLCs that contain ⬃800 –1,000
amino acids (147, 227, 299, 311). The NH2-terminal portion
of the vertebrate CLC proteins is embedded in the lipid
bilayer and forms the ion-transport pathway (77). The
large quantity of CLC proteins purified from E. coli renders it possible to grow two- and three-dimensional crystals for structural analyses (77, 78, 216). These structural
studies showed that a CLC functional unit consists of two
identical subunits, each containing an anion-transport
pathway related to each other by a twofold symmetry
perpendicular to the membrane plane (see Fig. 2A), con-
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ratio between the two transported substances. If n molecules of substrate S are transported in exchange of m
molecules of substrate C, and if the charge carried by
substrate S and C are ZS and ZC, respectively, the net
charge movement per transport cycle is nZS ⫺ mZC.
When this number is zero, the antiporter is not electrogenic, as in the Cl⫺-HCO⫺
3 exchanger in red blood cells
(95). In the cardiac Na⫹-Ca2⫹ exchanger, it has been
shown that three Na⫹ were transported in exchange for
the transport of one Ca2⫹ in the opposite direction (38).
Thus nZNa ⫺ mZCa ⫽ 3 ⫺ 2 ⫽ 1, making this transport
mechanism electrogenic. Because the transporter molecules in CLC family move Cl⫺ and H⫹ in opposite directions, the Cl⫺-H⫹ antiporter activity is always an electrogenic process regardless of the exchange ratio.
CFTR and CLC-0 are bona fide Cl⫺ channels because
⫺
Cl moves through the pore of these two molecules passively with a rate of 106–107 /s. Thus single-channel currents can be readily measured. Functional properties of
these Cl⫺ channels, however, suggest that they may be
viewed as evolutionary descendants of those transporters
in the ABC and CLC protein families. For example, hydrolysis of ATP by CFTR is coupled to a closed-openclosed gating cycle (97), and the opening of the CLC
channels is affected by Cl⫺ and H⫹ (51, 211, 240). In the
following discussions, we will first inspect the structure
and function of CLC and CFTR Cl⫺ channels and those of
their transporter counterparts. At the end of the paper, we
will speculate on the potential evolutionary linkage between ion channels and transporters in these two families.
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CLC-0 AND CFTR
firming the dimeric architecture implied from numerous
functional studies on CLC-0 in two decades (25, 174, 195,
196, 209, 210, 212, 213, 332). Within each subunit the
protein is made up of 18 ␣-helices, which are named helix
A to R, from the NH2 to COOH terminus of the protein.
Some of these ␣-helices run halfway through the lipid
membrane, with the positively charged end of the helix
dipole coordinating Cl⫺ in the middle of the cell membrane (77, 78). In the crystal structure, two anion-binding
sites are identified in the ion-transport pathway of the
wild-type protein. They are, respectively, named Scen and
Sint to reflect their locations (central, internal) in the pore
(Fig. 2A). The Cl⫺ at Scen interacts with the side-chain
hydroxyl of Ser-107 in helix D and that of Tyr-445 in helix
R, and also the main-chain nitrogen atoms from Ile-356
and Phe-357 of the helix N (77, 78). The Cl⫺-binding site
Sint is located ⬃6 –7 Å intracellular to Scen, approximately
at the junction where the intracellular aqueous solution
meets the pore. The Cl⫺ at this position is coordinated by
the backbone amide group from Gly-106 and Ser-107.
Even though Cl⫺ at Sint may appear to contact water
molecules on the intracellular side, the bound Cl⫺ at Scen
is hardly visible when the molecule is viewed from the
intracellular entrance of the Cl⫺-transport pathway,
mostly due to a constriction of the pathway near Ser-107.
However, a cysteine introduced at Tyr-445 in CLC-ec1 or
at the corresponding Tyr-512 position in CLC-0 is accessible to bulky methane thiosulfonate reagents applied
from the intracellular side (175, 205, 353), suggesting that
the constriction near Ser-107 does not completely obstruct the ion-transport pathway.
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The ion-transport pathway in the bacterial CLC
molecules is also constricted on the extracellular end
by the NH2 termini of helix F and helix N. A glutamate
residue (Glu-148) projects its side chain into the pore,
and the Cl⫺ exit pathway is occluded by this negatively
charged side chain at a location ⬃4 Å external to Scen
(Fig. 2A). When this glutamate is mutated to a noncharged amino acid, a Cl⫺ is seen at this position
(named Sext because this ion-binding site is external to
Scen) (78). This negatively charged glutamate residue is
conserved throughout the CLC family members except
for CLC-K. As will be described later, the glutamate
residue is critical for the transporter function of the
bacterial CLC molecules. The corresponding glutamate
residue also plays an important role in the gating properties of CLC channels.
The bacterial CLC structure reveals that Cl⫺ at these
three binding sites do not make direct contact with positively charged amino acids. Rather, they may be stabilized by the positive end (NH2 terminus) of helix dipoles
and by favorable, long-range, electrostatic interactions
(92). Whether all three binding sites can be simultaneously occupied by anions was recently addressed by
Lobet and Dutzler (185) by titrating the anion concentrations used in growing the bacterial CLC protein crystals
whose structures were later solved. Their data suggested
that all three binding sites can be occupied simultaneously by anions (185).
In summary, the CLC-ec1 structure shows multiple
Cl⫺-binding sites, and the transport pathway appears to
be disrupted by the side chain of Glu-148 and Ser-107 at
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FIG. 2. Structural models of CLC proteins. A: X-ray structure of CLC-ec1. Except for the four labeled amino acids in the subunit on left, only
backbone carbons of the molecule are presented. Green spheres are the two Cl⫺ seen in the crystal structure of the wild-type protein. They are
located at Scen and Sint as depicted. The side chains of Ser-107 and Y445 coordinate the Cl⫺ at Scen. The binding site Sext corresponds to the position
of the side chain of Glu-148, which is also the protonation site facing the extracellular medium (Gluex). The internal protonation site (Gluin) is
thought to be Glu-203. The inward and outward arrows depict the possible Cl⫺ and H⫹ transport pathways, respectively. B: potential structural
model of CLC-0. The membrane embedded portion is from the structure of CLC-ec1 (78), while the cytoplasmic portion is constructed by manually
assembling the two COOH termini of CLC-0 (208) in twofold symmetry. The two subunits are shown in blue and green, respectively. Residues in
red in the membrane embedded part represent the critical glutamate residue (Glu-166 of CLC-0), whose side chain is thought to be the fast-gate. The
curved arrows in red roughly represent the two independent transport pathways of Cl⫺. The arrows in deep blue represent the potential H⫹ pathway
as suggested from the experiments in CLC-ec1 (5). However, it is not known if a similar H⫹ pathway exists in CLC-0 (345).
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TSUNG-YU CHEN AND TZYH-CHANG HWANG
the extracellular and intracellular end of the transport
pathway, respectively. Because the structure of the
wild-type protein did not show an apparent open pore, it
was speculated that the structure might represent a closedstate channel. With hindsight, however, the multiple constrictions in the ion-transport pathway may have already
suggested that this bacterial molecule does not contain a
freely diffusible pathway for Cl⫺ and is likely to be a transporter. More recent functional studies, as described below, indeed provide evidence that the bacterial CLC proteins are Cl⫺-H⫹ antiporters.
B. Mechanistic Function of CLC-ec1
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The bacterial CLC molecules were discovered in the
1990s. The only functional study prior to the crystallographic era was performed using a flux measurement.
With this crude assay, and several biochemical approaches, Maduke et al. (198) first revealed the homodimeric arrangement of the two subunits and the preference for various anions in the E. coli CLC protein.
Under the false assertion that the bacterial CLC was an
ion channel, the anion preference was thought to be the
channel permeability sequence, which is roughly similar
to those of vertebrate CLC channels. Recently, a more
rigorous functional assay, the lipid bilayer recording, was
performed on E. coli CLC molecules (1, 3). These electrophysiological measurements showed that the reversal
potential of the current is determined by both the Cl⫺ and
H⫹ gradients across the membrane and that a diffusion
model based on the conventional Goldman-Hodgkin-Katz
equation cannot explain the experimentally determined
reversal potential. On the other hand, the calculation
based on a Cl⫺-H⫹ antiporter model with a coupling
transport ratio of 2Cl⫺:1H⫹ closely predicted the experimental results. It was thus proposed that the bacterial
CLC was a Cl⫺-H⫹ antiporter but not an ion channel (3).
The discovery that CLC-ec1 is a Cl⫺-H⫹ antiporter
immediately raised an interesting question: do vertebrate
or mammalian CLCs also function as transporters? For
nearly two decades, it was thought that the recorded
currents from eukaryotic CLC proteins were carried by
Cl⫺ through ion-diffusion pores. In the muscle-type CLC
channels (such as CLC-0 and CLC-1), the reversal potentials measured from these channels follow faithfully the
imposed Cl⫺ gradients, and are not altered by external or
internal pH (47, 116, 268, 269). In addition, these channels
showed sizable single-channel conductance, from ⬃1 to
10 pS, in physiological Cl⫺ solutions. Conductance in this
range corresponds to ⬃106–107 ions/s turnover in the pore
with a physiologically pertinent driving force, values too
large for the slow turnover rate of a transporter. However,
it was unclear whether members of the other two subbranches of CLC family might function as transporters.
Except for the muscle-type CLC channels, mammalian
CLC proteins are known to reside in the membrane of
intracellular organelles. By overexpressing these proteins
in heterologous expression systems, they can appear on
the plasma membrane. However, so far, no convincing
single-channel recordings have been obtained in these
members (cf. Ref. 330). Does the current generated from
these mammalian CLCs result from the Cl⫺-H⫹ antiporter
activity? Using H⫹-sensitive microelectrodes, Picollo and
Pusch (232) showed that the pH on the extracellular side
of the membrane was changed upon activation of CLC-4
and CLC-5, while the same assay failed to detect pH
changes when CLC-0 and CLC-2 were over-expressed.
Jentsch’s group (275) employed H⫹-sensitive dyes to detect the intracellular pH change using imaging methods.
They reached a similar conclusion that some mammalian
CLCs, including CLC-4 and CLC-5, indeed are able to
transfer protons across the cell membrane. The ability of
these mammalian CLCs to transport protons may well
explain their functions in the regulation of the intravesicular pH (114, 161, 171, 217, 300), a physiological role
similar to that played by bacterial CLC proteins in maintaining the normal pH within E. coli (140). Interestingly, a
CLC member from Arabidopsis thaliana CLCa (AtCLCa)
is able to accumulate nitrate in the vacuole, and this CLC
⫹
molecule behaves as an NO⫺
exchanger (67). The
3 -H
antiporter mechanism of this plant CLC protein is directly
connected with its physiological role for nitrogen fixation.
Thus it seems that both animals and plants have borrowed
the functional architecture of the primordial CLC protein
to perform anion-H⫹ exchange reactions.
The structural basis of the Cl⫺-H⫹ antiporter function has attracted attention ever since the bacterial CLC
molecule was known to be a transporter. Because of the
intriguing structural implication, the functional role of the
key glutamate residue, Glu-148, was examined first. Using
lipid bilayer recordings, Accardi and colleagues (1, 3)
showed that the coupled transporter behavior described
above disappeared when this glutamate residue facing the
extracellular side (Gluex) was mutated to alanine. In the
E148A mutant, H⫹ was hardly transported through the mutant protein, and the reversal potential of the mutated
CLC-ec1 closely followed the equilibrium potential of Cl⫺
calculated from the Nernst equation. Thus the mutant
protein became a Cl⫺ channel or a Cl⫺ uniporter. Accardi
et al. (5) went further to identify Glu-203 of CLC-ec1 as a
H⫹-binding site facing the intracellular medium (Gluin)
(5). Gluin is conserved among CLC family members in an
interesting way. In CLC members that are thought to be
Cl⫺-H⫹ antiporter, the corresponding residue is always a
glutamate. For those members that are thought to be Cl⫺
channels, the negatively charged glutamate is not conserved. These studies suggest that Glu-203 and Glu-148
are two protonation sites in the H⫹ transport pathway.
The Cl⫺ transport in CLC-ec1 likely follows the path-
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CLC-0 AND CFTR
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mechanism of Cl⫺-H⫹ antiporter mechanism in the bacterial CLC protein.
V. CLC-0: A PROTOTYPE CHANNEL IN THE
CLC FAMILY
A. Introduction
CLC-0 consists of two identical subunits, each containing 804 amino acids (209, 210). Identical channel behaviors can be obtained from patch-clamp recordings of
the expressed channels or from bilayer recordings of the
purified channel protein (209, 210), suggesting that no
auxiliary subunits are needed to generate functional
CLC-0 channels. The primary amino acid sequence of all
CLC molecules of vertebrate origin shows a molecular
mass of approximately twofold that of the bacterial CLC
proteins. The extra 400 –500 amino acids form the large
cytoplasmic domain. Thus the NH2-terminal half of vertebrate CLC proteins is membrane-embedded and forms
ion-transport pathways. Several mutational studies as
well as experiments using substituted cysteine accessibility method (SCAM) have indicated that the overall structure of the pore-lining portion of CLC-0 should be similar
to the anion transport pathway of bacterial CLC proteins
(48, 78, 81, 174, 175, 196). The crystal structure of the
COOH-terminal half of CLC-0, a component absent in
bacterial CLC structure, was recently solved by Meyer
and Dutzler (208). This COOH-terminal cytoplasmic domain contains a folded core of two tightly interacting
cystathionine ␤-synthetase (CBS) subdomains, and this
COOH-terminal domain from each subunit may form a
dimer (208). If the assembly of the NH2-terminal and the
COOH-terminal halves of CLC-0 does not distort the individual structure, the overall CLC-0 structure may be similar to that shown in Figure 2B (208). This overall structural architecture may also represent the structures of
other vertebrate CLC proteins.
Although various CLC Cl⫺ channels may have a similar structure, they exhibit very different functional properties. CLC-0 and CLC-1 are present in the muscle-related
tissues. CLC-1 is the principal Cl⫺ channel that contributes to a major part of the resting conductance of the
mammalian skeletal muscle, while CLC-0 exists in the
Torpedo electric organ, an organ that derived evolutionarily from skeletal muscles. These two Cl⫺ channels control the membrane potential critical for the excitability of
the cells (muscle cells and Torpedo electroplax, respectively). Therefore, depolarization-activated opening is important for their physiological roles, to repolarize the
membrane potential after excitation. On the other hand,
CLC-Ka and CLC-Kb are expressed on the cell membrane
of renal tubular cells to control the electrolyte balance
(293, 317), and in the organ of Corti to regulate the ion
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way defined by the three observed Cl⫺-binding sites in
the crystal structure. Thus the Cl⫺- and H⫹-transport
pathways of CLC-ec1 converge at the extracellular side,
but diverge at the intracellular side, as depicted in
Figure 2A (5).
One question arises with the identification of Glu148 and Glu-203 being the protonation sites in the H⫹
transport pathway of CLC-ec1: How is H⫹ transported
between these two sites? An examination of the CLCec1 structure suggests that the conserved residue Tyr445, whose side chain directly coordinates the Cl⫺ at
Scen, may be a midway station for H⫹ transport because
this residue is located halfway between Glu-148 and
Glu-203, and because numerous examples of tyrosine
hydroxyl groups are known to participate in H⫹ transfer in various proteins (137, 173). Accordingly, Accardi
et al. (2) mutated this conserved tyrosine residue and
found that the H⫹ transfer ability of CLC-ec1 was not
lost in Y445F or Y445W mutants, indicating that the
protonation of the tyrosine hydroxyl is not required for
the H⫹ transport. In the same series of experiments,
however, they found a striking correlation between the
anion occupancy at Scen and the Cl⫺-H⫹ coupled transport by replacing Tyr-445 with various amino acids. An
independent experimental maneuver, in which the coupled transport of H⫹ with various anions in the wildtype CLC-ec1 was measured, also revealed a similar
correlation between the anion occupancy at Scen and
the coupled transport (223). In light of these results,
Miller and colleagues proposed that the central Cl⫺
itself provides a transient protonation site necessary
for coupling H⫹ and Cl⫺ in the transport cycle (2, 223).
It has been suggested from a theoretical study that the
anion occupancy of the Cl⫺-binding sites in CLC-ec1
renders the intrinsic potential of Scen more negative
(345), a condition that may be required for H⫹ to pass
the energy barrier in the transport pathway. The proposal that the Cl⫺ itself provides a transient protonation site is still amazing because HCl is an extremely
strong acid in aqueous solutions. However, the low
dielectric constant inside a protein, and the pore electrostatic potential from many surrounding charged residues, may make this hypothesis tenable as argued by
Miller and colleagues (2).
That H⫹ transport requires anion occupancy at Scen
may suggest the synergistic binding of the transported
H⫹ and anions at around Tyr-445. The Cl⫺ and H⫹
transport pathways diverge from this coupling site toward the intracellular medium. Such a transport mechanism appears to be very different from the alternatingsite antiport mechanism described in Figure 1C, because no synergism between the bindings of two
substrates is allowed in the classical antiport model.
Further studies will be necessary to reveal the detailed
358
TSUNG-YU CHEN AND TZYH-CHANG HWANG
B. Channel Conductance and Ion Permeation
of CLC-0
Eukaryotic CLC channels are relatively nonselective among various monovalent anions: Cl⫺, Br⫺, NO⫺
3 ,
or even larger anions such as I⫺ and SCN⫺ are permeant (197). The permeability ratios of these anions
are usually within a factor of 10 (87, 90, 122, 193, 270,
353), with a relative permeability sequence: SCN⫺ ⬃
⫺
⫺
⫺
NO⫺
3 ⬎ Cl ⬎ Br ⬎ I . Measuring the single-channel
conductance of CLC channels has not been an easy task
because of the low conductance of most of CLC channels (197). Fortunately, CLC-0 has a conductance of
⬃10 pS in physiological Cl⫺ concentrations, a conductance large enough for single-channel recording experiments. In comparison, the single-channel conductance
values for CLC-1 and CLC-2 are, respectively, approximately eight- and threefold smaller than that of CLC-0
(245, 274, 332), making the single-channel studies of
these two mammalian channels quite challenging. For
molecules that are thought to be Cl⫺-H⫹ antiporters,
such as CLC-4 and -5, no convincing single-channel
traces have yet been obtained, consistent with the idea
that they may function as transporters.
Although the ion-permeation pathway of various
CLC channels may be similar to that suggested from the
bacterial CLC protein structure, the ion permeation
mechanisms in different channels exhibit different feaPhysiol Rev • VOL
tures. For example, the single-channel current-voltage
(I-V) relationship of CLC-0 is linear (176, 209, 210), but
that of CLC-1 is known to be inwardly rectifying as
deduced from the instantaneous I-V curve (87, 90, 122,
193, 270). Another more extreme example is the difference between CLC-0 and CLC-ec1. The critical residues
lining the ion-transport pathway are conserved between these two molecules (81, 86, 175), yet the former
is a Cl⫺ channel, while the latter is a transporter. The
structural determinants in CLC-ec1 that prevent the
Cl⫺-transport pathway from becoming a Cl⫺-diffusion
pathway is not yet known.
CLC-0 is an ion channel, so the ion-transport pathway
is open as an ion-diffusion pore as long as the side chain
of Glu-166 (which corresponds to Glu-148 or Gluexof CLCec1, and is probably the only gate in CLC-0) moves out of
the pore. Several theoretical calculations have been conducted to understand the ion permeation mechanisms of
CLC channels, using the atomic coordinates from bacterial CLC proteins (56, 57, 92, 215, 345). One study suggested a potentially important role of residue 318 of bacterial CLCs, which corresponds to G352 and E417 in
CLC-0 and CLC-1, respectively. Brownian dynamic simulations showed that the charge at this position may determine the Cl⫺ exit rate towards the extracellular solution,
thus explaining the differences between CLC-0 and CLC-1
in single-channel conductance as well as in the rectification property of I-V curves (57). Another study calculated
the source of the stabilizing energy of the bound Cl⫺ and
predicted that the positive charge of the residues Lys-131
and Arg-340 (corresponding to residues 149 and 401 of
CLC-0) stabilizes Cl⫺ binding in the ion-transport pathway
(92). These computational studies have a major limitation
in that the bacterial CLC molecules are Cl⫺-H⫹ antiporters but not ion channels. Because the fundamental principles of ion transport between ion channels and transporters are so different, it is not known if any conclusions
from these computational studies of bacterial CLC proteins are indeed valid for CLC channels. To examine the
theoretical predictions, Zhang et al. (353) tested the functional importance of the corresponding charged residues
in CLC-0 and CLC-1. The results suggested that some (e.g.,
Lys-149 of CLC-0 corresponding to Lys-131 of CLC-ec1)
but not all of the predicted residues assume a functional
role in determining the pore functions of CLC-0. The
results, however, do not rule out potential functional roles
of these residues in the transporter function. The role of
Lys-149 in CLC-0 functions was further elaborated by the
recent work of Engh et al. (80).
Besides residues suggested from theoretical calculations, mutagenesis approaches for more than a decade
have identified several important charged residues that
contribute electrostatic energy to the pore (48, 174, 209).
Being an anion channel, the ion-binding sites in the CLC-0
pore must be associated with an intrinsic positive poten-
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transport across the stria vascularis (84, 247). Thus a
continuous opening of such a CLC channel to support the
transepithelial Cl⫺ transport may be more suitable for
their primary physiological roles. Compared with the limited expression of other CLC channels, CLC-2 is widely
distributed in almost all tissues, including neuronal and
cardiac tissues, and this channel is opened by membrane
hyperpolarization. A physiological role of CLC-2 in mediating the paradoxical excitation produced by GABA receptor activation in selected neuronal populations has
been suggested (55, 297, 298).
Among these CLC Cl⫺ channels, only the gating function of CLC-0 has been studied in detail, and the properties potentially related to transporter functions (i.e., the
regulation of gating by Cl⫺ and H⫹ ions) have been well
documented. Although still speculative, comparing the
structure-function relation between CLC Cl⫺ channels
and CLC Cl⫺-H⫹ antiporters may provide the opportunity
to understand the evolutionary link between the two categories of protein functions in the CLC family. In the
following section, we will only discuss the structure-function relationship of CLC-0, the prototype Cl⫺ channel in
the CLC family. Readers who are interested in CLC-1,
CLC-2, and CLC-K channels are referred to several review
articles (18, 51, 146, 317).
359
CLC-0 AND CFTR
tial, a feature that has been experimentally revealed by
modifying the introduced cysteine using charged methane
thiosulfonate reagents (175). The positive intrinsic potential in the pore of CLC-0 might help select anions versus
cations. The same positive intrinsic potential then disfavors the movement of H⫹ to the pore to protonate the side
chain of Glu-166, a process thought to be critical for the
gating mechanism of CLC-0. Therefore, the occupancy of
Cl⫺ may be important for the protonation of pore residues, as suggested by theoretical calculations. The computational study also suggested several potential pathways for H⫹ permeation through the bacterial CLC protein, which may or may not exist in CLC-0 (345).
The gating of CLC-0 consists of multiple mechanisms,
which are summarized in Figure 3. Based on the kinetics
of the channel opening, the gating of CLC-0 includes “fast”
FIG. 3. Gating functions of CLC-0. A: single-channel behaviors of CLC-0. Top panel shows a 14-s recording for CLC-0 at ⫺60 mV under a
symmetrical Cl⫺ concentration of 120 mM. The dotted line represents the zero-current level. The recording was obtained from an inside-out
membrane patch excised from a Xenopus oocyte expressing wild-type CLC-0. Arrows indicate the long inactivation events corresponding to the
closure of the slow-gate. The activities between the inactivation events represent the fast-gating transitions, which can be seen better with the
expanded trace on the left. The fast-gate activity fluctuates among three equidistant current levels, and the probabilities of these three current levels
follow a binomial distribution. The expanded trace on the right shows a transition from the slow-gate, open state to the slow-gate, closed state.
B: fast-gating at the macroscopic current level. The recording was made from the excised inside-out membrane patch of the human embryonic
kidney (HEK) 293 cell expressing the inactivation-suppressed mutant C212S. The prepulse voltage is 100 mV, while the tail voltage is ⫺100 mV. The
tested voltage is from ⫹100 to ⫺160 mV in ⫺20 mV steps. Notice that the time constant of the current relaxations upon voltage change is ⬃10 ms.
C: the slow-gating process of the wild-type CLC-0 examined by macroscopic current recordings from an excised inside-out membrane patch. Left
panel shows original recording traces under voltage clamp from the holding voltage of 0 mV to ⫹40 mV for 50 ms. One pulse was given every 6 s
to monitor the relatively slow time course as shown in the right panel (temperature ⫽ 23.5°C). Circles in the right panel represent the current
measured from the end of the voltage pulse (averaged from the traces between two vertical lines in the left panel). For B and C, the patches were
recorded in symmetrical 140 mM Cl⫺ solutions, and dotted lines represent the zero-current level.
Physiol Rev • VOL
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C. Gating Properties of CLC-0
and “slow” gating, which operate in the time scale of
milliseconds and seconds, respectively. Shown in Figure
3A is a single-channel recording trace of CLC-0. The long,
nonconducting states between bursts of channel activities
(indicated by arrows in Fig. 3A) represent the closure of
the slow-gate. When the slow-gate is open, a burst of
channel activity appears, and three current levels (the
nonconducting level, the middle level, and the fully open
level) can be observed. The current fluctuation within a
burst is called fast-gating because the gating mechanism
operates at a much faster time scale (milliseconds) than
that of slow-gating (seconds to minutes). Relaxation of
the macroscopic current of CLC-0 provides ways to measure the fast-gating and the slow-gating kinetics from
ensemble of channels (Fig. 3, B and C).
The characteristics of fast-gating in single-channel
recordings, three equally spaced current levels and the
binomial distribution of the probabilities of these three
current states, have been observed for more than two
360
TSUNG-YU CHEN AND TZYH-CHANG HWANG
1. Slow-gating function of CLC-0
Slow-gating of CLC-0 is controlled by membrane potential. Membrane depolarization favors the nonconducting state of the slow-gate; the closed state of the slow-gate
at depolarized potential normally lasts for seconds or tens
of seconds, mimicking the inactivation of the voltagegated cation channels. Thus slow-gating of CLC-0 is sometimes called “inactivation gating.” In addition to membrane potential, slow-gating of CLC-0 is also controlled by
temperature, chloride ion concentration, and pH. High
extracellular pH, low extracellular Cl⫺, and high temperature favor a nonconducting (inactivation) state of the
slow-gate (47, 50, 53, 214, 243, 336). A recent study suggests that slow-gating may also be modulated by the
redox state of the environment (172). These studies, however, have not provided mechanistic information regarding the molecular mechanism of slow-gating. An experiment in the early 1990s showed an interesting phenomenon that the operation of the slow-gate is through a
nonequilibrium gating cycle, in which the closure of the
slow-gate predominantly succeeds a state in which one of
the two fast-gates is open, while the slow-gate opens to a
state in which the fast-gates of both pores are open (262).
This nonequilibrium gating is more prominent with a
larger Cl⫺ gradient across the channel pore imposed by
the membrane voltage (262). Recent unpublished data,
however, suggest that it may be the H⫹ gradient across
the cell membrane that provides the energy for the
nonequilibrium gating cycle (M. Maduke, personal communication). From a retrospective view, although the
mechanisms underlying these effects are not known,
the nonequilibrium gating cycle suggests that the CLC-0
slow-gating may be coupled to the fast-gating, and the
gating mechanisms are linked to the Cl⫺/H⫹ gradients
across the cell membrane. However, without knowing the
Physiol Rev • VOL
physical nature of the slow-gate, the coupling mechanism
remains elusive.
Electrophysiological measurements have shown that
the open probability (Po) of the slow-gate is reduced upon
increasing temperature. Because this temperature dependence is extremely high, with a Q10 of ⬃40 (50, 243), a
large protein conformational change is thought to be associated with the slow-gating transition. Extracellular
Zn2⫹ are known to facilitate the inactivation process, thus
inhibiting CLC-0 (50). The effect of extracellular Zn2⫹ on
the slow-gating process is supported by the observation
that a mutant C212S, whose slow-gate is locked in the
open state, loses its sensitivity to Zn2⫹ inhibition (176).
Although the slow-gating of CLC-0 can be affected by
numerous point mutations, including C213G, C480S (176),
P522G, and L524I (243), the mechanism underlying the
mutational effects is largely unknown. Manipulations
that disrupt the COOH-terminal intracellular domain
have been shown to affect the kinetics of slow-gating,
suggesting that, in addition to the membrane-spanning
part of the protein, the COOH-terminal half of the channel is also important for slow-gating (85, 94, 120, 121,
132, 199, 277, 342).
Sequence comparisons and a recent crystallographic
study of the COOH-terminal domain of CLC-0 have revealed that ATP-binding CBS domains exist in the COOH
terminus of CLC channels (85, 203, 208). The binding of
ATP to the COOH terminus of CLC-1 has been demonstrated to affect channel gating (28). One observation
from the crystallographic study of CLC-0 is that the two
COOH-terminal domains of CLC-0 form a dimer in solutions, but the dimeric structure is not present when the
proteins form a crystal (208). Thus the strength of this
dimeric interaction is probably weak, raising the possibility that the interaction of the two COOH termini may be
dynamically associated with channel gating. Recent experiments using fluorescence resonance energy transfer
(FRET) techniques showed that the COOH terminus of
the two CLC-0 subunits may undergo a large movement
relative to each other (39), reminiscent of the speculation
by Miller more than two decades ago (213). Experiments
in CLC-2 and C. elegans CLC channels also showed that
point mutants, deletion mutants, or splice variants of the
cytoplasmic COOH terminus alter not only the slow-gating but also the accessibility of the pore to extracellularly
applied methane thiosulfonate reagents (70, 120, 347).
These results may suggest that alterations in the cytoplasmic domain lead to a conformational change of the outer
pore vestibule and the associated glutamate gate. Perhaps
the relative movement of the COOH-terminal domains
(11) is transduced to the two Cl⫺-permeation pores
through the last alpha helix (helix R) of the transmembrane domains, because Cl⫺- and pH-dependent fluorescence changes from fluorophores labeled at helix R of
CLC-ec1 have been observed, suggesting a gating-associ-
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decades. Such a unique property prompted Miller and
co-workers (116, 212, 213) to argue that a single CLC-0
channel contains two ion-permeation pores. The three
equidistant current levels with binomial distribution are also
found in CLC-1 (274). In addition, when heterodimeric channels were generated by coexpressing wild-type and mutant CLC-0 channel subunits, or by linking subunits of
CLC-0 and CLC-1, CLC-0 and CLC-2, or CLC-1 and CLC-2,
single channels with two nonidentical but independent
pores were observed (174, 196, 209, 332). Thus the “double-barreled” model of CLC channels had been firmly
established even before the crystallographic era. From
the now available high-resolution bacterial CLC structures, CLC-0 should consist of a membrane portion that
forms the ion-conducting pore linked with a COOH-terminal cytoplasmic portion whose structure has also been
solved recently (208).
361
CLC-0 AND CFTR
ated movement of this helix (27). However, direct structural studies of CLC-ec1 offer no such conformational
change for helix R in various Cl⫺ and pH conditions (185).
Thus a unified picture in the gating-associated conformational change of CLC channels has not yet been settled.
2. The fast-gate opening mechanism of CLC-0
Physiol Rev • VOL
D. Mechanistic Models for CLC-0 Fast-Gating
In the last decade, several mechanistic models have
been developed for the fast-gating mechanism of CLC-0.
In the first class that could be called “conventional” theory, the fast-gate of CLC-0 was proposed to be opened by
extracellular Cl⫺. The idea first came from an observation
that Cl⫺ and various permeable anions can shift the Po-V
curve of the CLC-0 fast-gate (244). In the original model,
it was proposed that the gate is linked with an anionbinding site in the pore, and the opening of the fast-gate
may come from a direct activation by the binding of
extracellular Cl⫺ to this binding site located within the
membrane electric field. Because Cl⫺ carries a negative
charge, the voltage dependence of the fast-gate opening
could then come from the voltage-dependent binding step
(244). A slightly modified model was later proposed by
suggesting that Cl⫺ binding to the pore is not a voltagedependent process (53; but see Ref. 79). Rather, Cl⫺ may
first bind to a site outside the membrane electric field, and
the Cl⫺-bound channel then undergoes a conformational
change, which translocates the bound Cl⫺ to an inner site
within the membrane electric field and opens the gate (53;
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In single-channel recordings, the transitions within
the burst represent the fast-gating activities of CLC-0. The
opening of this gate depends on the membrane potential
and is regulated by pH and Cl⫺. At physiological pH and
Cl⫺ concentrations (for example, symmetrical 150 mM
Cl⫺ on both sides of the membrane), the fast-gate Po is
higher at more depolarized membrane potentials (53, 116,
176, 210, 212, 213, 244). For example, at ⫺120, ⫺90, and
⫺60 mV, the fast-gate Po is ⬃0.3, 0.5, and 0.7, respectively.
The slope of the Po-V curve corresponds to an e-fold
change in Po per ⬃25 mV, which, according to the conventional idea of voltage dependence, may imply that the
total gating charge movement is equivalent to the movement of one unit charge across the entire membrane
electric field.
Single-channel studies revealed that the slope of the
Po-V curve came from two sources: the voltage dependence of the fast-gate opening rate (which has a positive
slope with a gating charge of ⬃0.7) and that of the closing
rate (which has a negative slope with a gating charge of
⬃0.3) (53). Experimental evidence suggests that the opening and the closing processes of the fast-gate may not be
the reverse of each other and may have to be considered
separately. The positive slope of the voltage dependence
of the opening rate indicates that opening of the fast-gate
is favored by membrane depolarization. However, this is
only a simplified description of fast-gate opening. When
the membrane potential is hyperpolarized to a very negative range (for example, more negative than ⫺150 mV),
the depolarization-activated channel activity of CLC-0 is
very small, but the channel’s Po does not approach zero.
Instead, in this very hyperpolarized voltage range, the
fast-gate opening rate is paradoxically increased by membrane hyperpolarization (53). Thus, besides a depolarization-activated opening mechanism (in the more depolarized voltage range), there is a hyperpolarization-activated
opening mechanism for the fast-gate of CLC-0; the fastgate opening process may have multiple components with
opposite voltage dependence. On the other hand, the
closing rate appears to depend on the membrane voltage
monotonically, with a fixed slope of approximately ⫺0.3
throughout the voltage range that has been examined (47,
52, 53).
The control of fast-gate opening of CLC-0 by Cl⫺ and
pH provides a first glimpse for a possible relationship
between this gating process and the Cl⫺-H⫹ antiporter
activity. It is the depolarization-activated, fast-gate open-
ing that is regulated by [Cl⫺]o; reducing [Cl⫺]o shifts the
opening rate curve to the depolarization direction, thus
making the fast-gate more difficult to open. The closing
rate, however, is not significantly affected by altering
[Cl⫺]o (47, 52, 53). Therefore, the overall effect of reducing [Cl⫺]o is a parallel shift of the steady-state Po-V curve
of the fast-gate towards a more depolarized direction.
Such an effect can also be achieved by reducing the
intracellular proton concentration ([H⫹]i). As revealed by
Hanke and Miller (116) more than two decades ago, increasing the pH of the intracellular solution by 1 unit
(10-fold reduction in [H⫹]i) shifts the Po-V curve to the
right by ⬃60 mV. Thus altering [Cl⫺]o and [H⫹]i appears to
exert, at least at the phenomenological level, a similar
effect on the fast-gate Po-V curve.
On the other hand, the hyperpolarization-activated
fast-gate opening mechanism is not regulated by [Cl⫺]o or
[H⫹]i. It is the extracellular H⫹ ([H⫹]o) that helps activate
the fast-gate opening at the hyperpolarized membrane
potential (47). The overall effect of increasing [H⫹]o (or
reducing extracellular pH) is an elevation of the minimal
Po of the fast-gate Po-V (47, 269), an effect very different
from the shift of the Po-V curve along the voltage axis in
response to the change of [Cl⫺]o and [H⫹]i. Thus the
regulations of fast-gating by Cl⫺, H⫹, and membrane potential appear to be very complicated. A unified theory is
urgently needed to explain all these gating phenomena. A
successful model will promote our understanding of the
structure-function relationship of CLC-0 as well as the
linkage of CLC channels and transporters.
362
TSUNG-YU CHEN AND TZYH-CHANG HWANG
Physiol Rev • VOL
Based on the functional relationship between CLC-0 and
CLC-ec1, Miller (211) proposed a “degraded transporter”
mechanism for the fast-gating mechanism of CLC-0. In
this model, the side chain of Gluex (Glu-166 of CLC-0) is
the gate, and protonation of this negatively charged side
chain opens the gate. When the membrane potential depolarizes, H⫹ might reach Glu-166 by means of “transport”
from the intracellular side. Thus membrane depolarization or an increase of [H⫹]i favors the opening of the
fast-gate. The role of Cl⫺ in this model could then be
acting as an allosteric modulator. For example, Cl⫺ binding to the pore may permit the transport of H⫹ to reach
the side chain of Glu-166 (2, 223, 345).
The degraded transporter model for fast-gating of
CLC-0 can also explain the extracellular H⫹ effect. At very
negative membrane voltages, outward movement of intracellular H⫹ is not favored, but the inward movement of
H⫹ from the extracellular side to Glu-166 may instead
replace the outward H⫹ movement to open the fast-gate
(211). The transport of H⫹ from the intracellular and the
extracellular solutions to Glu-166 should be voltage dependent. The experimental observation that the depolarization-activated opening (due to movement of H⫹ from
intracellular solution to Glu-166) is associated with a
gating charge of 0.7, while the hyperpolarization-activated
opening (favored by external H⫹) has a gating charge of
⫺0.3 is consistent with such a picture. A recent functional
study of the E166D mutant provided further evidence that
both the external and internal H⫹ can reach this residue
(316). Thus, if H⫹ indeed can reach the side chain of Gluex
to control the fast-gate opening, H⫹ must be transported
across CLC-0. Unlike the traditional model in which Cl⫺ is
the activating ligand, this new model suggests that H⫹ is
the activating ligand, and the gating charge for the voltage-dependent opening of the CLC-0 fast-gate is H⫹ but
not Cl⫺ (211)! However, if H⫹ is solely responsible for the
gating charge, one might expect that the closing rate will
also be increased by the membrane depolarization if H⫹ is
transported from Gluex to the external solution during the
fast-gate closing process. This prediction on the closing
rate apparently contradicts the experimental observation.
The degraded transporter model does not require a
concerted exchange of H⫹ with Cl⫺, like the coupled
Cl⫺-H⫹ exchange shown in CLC-ec1. In essence, therefore, the model presents CLC-0 as an H⫹-gated Cl⫺ channel, with Cl⫺ being an allosteric modulator. A model that
more closely links the gating of CLC-0 with the Cl⫺-H⫹
antiporter activity in CLC-0 may also be possible; for
example, the opening and closing of the gate of CLC-0 is
strictly coupled to a Cl⫺-H⫹ antiporter activity such that
one transport cycle is associated with an open-close transition for channel activities. Such a model may be called
a “coupled transporter” model. In this case, the observed
current in CLC-0 may come from two parts: the ionic
current through the CLC-0 pore and the transporter cur-
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cf. Ref. 79). Though slightly different in their interpretations of voltage dependence, both models proposed that
Cl⫺ could be the activating ligand of the fast-gate, and an
inward movement of Cl⫺ in the membrane electric field
will provide the energy needed for a voltage-dependent
process, that is, the Cl⫺ is the gating charge of this voltage-dependent channel. These models thus suggested that
the voltage dependence of CLC-0 does not require a voltage sensor built in the channel molecule like that in the
S4-type cation channels (148, 149, 189, 190). The crystal
structure of the bacterial CLC proteins indeed revealed
that CLC-type proteins do not possess structural motifs
serving as voltage sensors (77, 78). The obstruction of the
Cl⫺-transport pathway by the side chain of Glu-148 in
CLC-ec1 was used to suggest that the side chain of the
corresponding glutamate residue in CLC-0, Glu-166, may
function as the fast-gate, and the competition of Cl⫺ with
the negatively charged side chain of the key glutamate
residue may be the mechanism underlying fast-gate opening. Such a “gating-permeation coupling” idea was recently further examined by a computational study using
metadynamic methodology, in which the main barrier to
the Cl⫺ translocation near Gluex was reduced by an ionpush-ion effect (102). Another computational study also
supports the depolarization- and hyperpolarization-activated fast-gating by showing the interaction of the side
chain of Gluex with external Cl⫺ and H⫹ (32). These
conventional models of fast-gate opening by Cl⫺, however, do not describe the functional role of intracellular
H⫹ in the fast-gate opening mechanism. Nevertheless, one
simple possibility might be that the protonation of the
channel reduces the free energy difference between the
closed state and the open state of the fast-gate, resulting
in a leftward shift of the Po-V curve. In this sense, H⫹
serves as an “allosteric modulator” for the opening of the
CLC-0 fast-gate, a speculation made when the internal pH
effect was first discovered (116).
In contrast to the idea that Cl⫺ serves as the activating ligand, a model was recently proposed by Miller (211)
in which the roles of Cl⫺ and H⫹ in the CLC-0 fast-gating
are transposed. This model was inspired by the recently
known structural/functional properties of CLC-ec1, such
as 1) removing the negative charge of Glu-148 in CLC-ec1
appears to make its side-chain swing out of the ion transport pathway, and that the mutation of the corresponding
Glu into a neutral residue converts eukaryotic CLC channels into a constitutively open channel; and 2) the coupling of Cl⫺ and H⫹ transport was disrupted by removing
the titrable group of Glu-148 (Gluex) and Glu-203 (Gluin),
indicating that a H⫹-transport pathway distinct from the
Cl⫺ pathway may exist in CLC proteins, a speculation also
suggested by a computational study (345). If the functional operations of CLC-0 retain some remnants of
Cl⫺-H⫹ antiporter properties, protonation of Glu-166 may
be the ultimate step that opens the fast-gate of CLC-0.
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CLC-0 AND CFTR
E. Unsettled Issues and Future Directions
Each of the aforementioned models describes a specific aspect of the fast-gating mechanism, but falls short in
explaining other gating properties. Overall, these models
may be grouped into two general categories: those which
propose that opening of the gate is via the competition
between Cl⫺ and the channel gate (Cl⫺ is the activating
ligand, the conventional models), and those that propose
that the opening of the gate is somewhat related to the
Cl⫺-H⫹ antiporter activity (H⫹ as the activating ligand,
the transporter model). In conventional models, the fastgate opening by Cl⫺ is thought to result from a mechanism whereby Cl⫺ occupancy at Sext repels the negatively
charged side chain of Gluex. However, single-channel
studies in CLC-0 have suggested that the competition
between Cl⫺ and the side chain of the glutamate residue
may be used to explain the effect of [Cl⫺]i on the closure
of fast-gate; fast-gate closure of CLC-0 is reduced by an
increase of [Cl⫺]i (49, 52–54), a “foot-in-the-door” effect
first described in cation channels (304). The internal and
external Cl⫺ effects on fast-gating of CLC-0 are very different. For example, altering [Cl⫺]o mainly affects the opening
rate, while changing [Cl⫺]i alters the closing rate but has
little effect on the opening rate of the fast-gate (52, 53).
The apparent Cl⫺ affinities in modulating the opening rate
and the closing rate by [Cl⫺]o and [Cl⫺]i, respectively,
differ by ⬎20-fold, suggesting that the Cl⫺-binding sites
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involved in the opening and closing mechanisms may be
different (52, 53). In addition, the fast-gate opening and
closing mechanisms are differentially affected by point
mutations. For mutations at the external pore entrance of
CLC-0, for example, K165, the effect is on the opening rate
but not the closing rate (174). On the other hand, for a
mutation at the intracellular pore entrance of CLC-0,
K519, the effect is more on the closing rate but not the
opening rate (52). Taken together, these results strongly
suggest that these two aspects of the fast-gating mechanism, the opening and closing processes, may not be
reversible processes of each other. If the foot-in-the-door
effect of Cl⫺ in the pore, that is, the competition of Cl⫺
with the negatively charged side chain of Glu-166 for
occupying Sext, is the mechanism underlying the Cl⫺ effect on the closing rate, it seems difficult to use the same
mechanism to explain the external Cl⫺ effect on the
fast-gate. In fact, it was questioned previously if the competition of Cl⫺ with the Glu-166 side chain could reflect
the fast-gate opening mechanism because the opening
rate and the closing rate of the fast-gate are separately
controlled by external and internal Cl⫺ in very different
ways (49).
The “transporter” model of the fast-gating mechanism is not without flaws. First, the strictly coupled transporter model appears to be less convincing because of a
dichotomy in the mutational results of the Tyr to Ala
mutation at the center of the transport pathway. In CLCec1, Y445A mutation significantly reduces the Cl⫺ occupancy at Scen and therefore severely uncouples the
Cl⫺-H⫹ transport (2). However, the corresponding mutation of Y512A in CLC-0 is almost without functional consequence (4). Thus, if a H⫹-transport pathway exists in
CLC-0, it must differ from that in CLC-ec1, and the Cl⫺
and H⫹ transports may not be tightly coupled, the essence
of the degraded transporter model. Even with the degraded transporter model, however, several problems remain unsettled. For example, how does Cl⫺ help H⫹ in
opening the fast-gate? Previous studies have shown that
only anions that are permeable through the pore or are
able to block the pore can increase the fast-gate opening
of CLC channels (244, 270). Thus the binding sites for
these anions to exert their effects on fast-gating likely
reside in the pore, and the occupancy of anions in the
pore must be critical for H⫹ to reach Gluex. However,
although computational studies suggest the presence of
such anion binding sites (92, 345), the crystal structure of
CLC-ec1 provides no evidence of anion-binding sites extracellular to the gate. Recent studies in CLC-ec1 showed
that the occupancy of an anion at Scen is critical for H⫹
transfer from Gluin to Gluex (223). However, Scen is intracellular to the side chain of Gluex. When the gate is closed,
how does a Cl⫺ in the extracellular medium reach Scen?
Another unknown in the transporter model is the
intracellular protonation site. If H⫹ is the activating ligand
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rent through a Cl⫺-H⫹ antiport cycle. However, this antiporter current would be difficult to detect by the reversal
potential measurement because the Cl⫺-H⫹ antiporter activity in CLC-0, if it exists, would be four to five orders of
magnitude smaller than the current carried by the Cl⫺ flux
through the CLC-0 pore.
The transporter models, especially the feature of H⫹
activation of CLC-0, help explain the variable gating phenomena among different CLC channels and their mutants.
As described above, the opening of CLC-2 is favored by
membrane hyperpolarization (108, 225, 242, 311, 320,
361). In addition, various manipulations of channel proteins, including single-point mutations, in CLC-0 and
CLC-1, convert the channel into a hyperpolarization-activated channel (88, 89, 194, 199, 277, 331, 352). It is interesting to point out that the hyperpolarization-activated
opening mechanism of CLC-2, and the artificially created
hyperpolarization-induced channel opening in CLC-0 and
CLC-1 mutants, are sensitive to external pH (242). Perhaps in hyperpolarization-induced channel gating, the H⫹
transport pathway from Gluin to Gluex is disrupted, and
therefore, the depolarization-activated opening mechanism is not functional, leaving only the hyperpolarizationactivated opening mechanism which results from protonation of Gluex by extracellular H⫹.
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TSUNG-YU CHEN AND TZYH-CHANG HWANG
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channel) is that the ligand binding is stoichiometrically
coupled to one closed-open-closed gating cycle. At this
point, however, without a firmly documented trans-channel movement of Cl⫺ or H⫹, the idea of an irreversible
gating cycle for CLC-0 channels, although conceptually
invigorating, remains somewhat hypothetical. In the following discussions, we will present CFTR, a Cl⫺ channel
gated by ATP. Being a member of the ABC transporter
family, CFTR is known to hydrolyze ATP (170). An inescapable inference here is an irreversible gating cycle
driven by the input of free energy from ATP hydrolysis.
From a wealth of structure-function studies of ABC transporters, we will try to catch a glimpse of the relationship
between ion channels and transporters using CFTR as a
model system.
VI. ABC TRANSPORTER FAMILY
The ABC transporters, one of the largest families of
homologous proteins, consist of integral membrane proteins
that share unique topological characteristics, namely, two
membrane-spanning domains (MSD) and two nucleotidebinding domains (NBD) (125, 127). Nearly all the members of the ABC superfamily perform active transport of
substrates against cellular concentration gradients, utilizing ATP hydrolysis as the source of the free energy. ABC
proteins are found in all prokaryotic and eukaryotic cells.
This gene superfamily is so prevalent in prokaryotic cells
that 3–5% of the bacterial genome sequenced to date
encodes ABC proteins (258). In the human genome, on the
other hand, 48 ABC transporters have been identified (68).
Based on phylogenetic analysis, the human ABC superfamily has been classified into seven subfamilies (ABCA–
ABCG). The importance of ABC proteins in human physiology is attested by the fact that 17 of the 48 human ABC
proteins are linked to genetic diseases. These include
Tangier disease (ABCA1) (159), Stargardt disease (ABCA4)
(157), Dubin-Johnson syndrome (ABCC2) (164), familial persistent hyperinsulinemic hypoglycemia of infancy (ABCC8)
(312), and cystic fibrosis (ABCC7) (263). In addition to
causing these diseases, multidrug resistance empowered by ABC transporters that export antibiotics or
chemotherapeutic reagents poses significant clinical
hurdles for the treatment of bacterial infection (165,
233) and cancer (13).
The functional diversity of members of this superfamily is suggested by the fact that human ABC proteins
have a wide tissue distribution (69). In addition, the substrates for different ABC proteins cover a broad spectrum
of substances, including sugars, amino acids, ions, drugs,
polysaccharides, and proteins. It seems logical to imagine
that once a primordial but effective “pumping machine”
employing the basic modular design of the ABC transporter (see sect. VII) emerged, more ABC proteins evolved
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of CLC-0, and if fast-gating of CLC-0 indeed bears the
lineament of the Cl⫺-H⫹ antiporter activity, one may expect that the protonatable residues are conserved. Gluex
is indeed conserved throughout the CLC family except for
CLC-K, a channel without prominent gating kinetics.
However, the intracellular Glu (Gluin) is conserved only in
those members that are thought to function as Cl⫺-H⫹
antiporters (5). In CLC-0, the residue corresponding to
Glu-203 of CLC-ec1 is a valine, a residue that cannot be
titrated by H⫹. If the H⫹ transport pathway exists in
CLC-0 to allow H⫹ movement from intracellular solution
to Gluex, where is the initial intracellular protonation site
responsible for the internal H⫹ regulation of CLC-0 fastgating? The degraded transporter model may argue that
the requirement of a conserved titratable residue facing
intracellular medium may not be necessary (211). However, a computational study suggested that the negative
potential near the residue corresponding to Glu-203 of
CLC-ec1 is absent in the CLC-0 homology model (345); the
lack of this negative potential would prevent proton transfer in this region. If this is the case, how does internal H⫹
reach to Gluex?
In all proposed gating mechanisms of CLC-0 described in this review, the interaction of Cl⫺ and/or H⫹
with the gate may provide the evolutionary link between
channels and transporters in this family. Detailed Cl⫺ and
H⫹ pathways and the Cl⫺- and H⫹-binding sites on the
CLC-0 channel are also critical for understanding the
gating mechanisms. Therefore, identification of the internal protonation site(s) responsible for the internal pH
effect on fast-gating should be an important problem to
tackle in the future. The CLC-ec1 structure suggests a
Cl⫺-permeation pathway that is evolutionarily preserved
in CLC-0, but whether there is a similar H⫹ transport
pathway in CLC-0 remains an outstanding question. The
conventional models propose that the gate is opened by
Cl⫺. Where Cl⫺ binds in the pore before it opens the gate
is still unsettled. All these questions are simply asking:
where are the binding sites for the activating ligands in
CLC-0, and where do these ligands go after they interact
with those sites? Finally, although the major portion of
the above discussion centers on the relation between the
antiporter activity and the fast-gating of CLC-0, the relation of the antiporter activity with the operation of the
CLC-0 slow-gating may also need to be contemplated in
light of the reports that the nonequilibrium slow-gating
cycles are fueled by the Cl⫺ gradient (262), and perhaps
the H⫹ gradient (M. Maduke, personal communication)
across cell membranes.
As mentioned at the beginning, CLC-0 can be viewed
as a ligand-gated channel, and the biophysical studies
have suggested that the ligands may be Cl⫺ and/or H⫹.
The special feature that is thought to distinguish this
channel from the classical neurotransmitter-gated channel (e.g., acetylcholine receptor or the GABA-gated Cl⫺
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CLC-0 AND CFTR
VII. STRUCTURE/FUNCTION OF ABC
TRANSPORTERS
A. Structural Characteristics of the Nucleotide
Binding Domain in ABC Transporters
The core domains (2 MSDs and 2 NBDs) of eukaryotic ABC transporters are usually encoded by a single
gene. Thus a single polypeptide contains these four core
domains. For many of the prokaryotic ABC proteins, however, individual domains are encoded by different genes,
and the resulting polypeptides are assembled into multidomain proteins. Among different members of the ABC
superfamily, the MSDs are highly divergent in both amino
acid sequence and topology, whereas the NBDs share
significant sequence and structural homology. This is perhaps not surprising since the MSDs likely contain substrate binding sites that must be different in different
transporters. In addition, while the NBDs serve as the
common engine to harvest the free energy of ATP hydrolysis, the MSDs will perform different functions depending on the physiological roles a particular ABC transporter assumes.
Like other ATP binding proteins, the ABC transporters’
NBDs contain the characteristic Walker A (GxxGxGKS/T, x
represents any amino acid) and Walker B (hhhhD, h represents a hydrophobic amino acid) nucleotide binding
motifs (327). Unique to the ABC superfamily is the “signature sequence” (LSGGQ) whose function remains unclear in spite of early speculation that it serves to transduce signals from ATP binding/hydrolysis to the conformational changes in other domains of the ABC
Physiol Rev • VOL
transporter during a transport cycle (14). In 1998, Hung
et al. (135) reported the first crystal structure of the
ATP-NBD complex from the histidine permease (HisP) of
Salmonella typhimurium.
As seen in other nucleotide binding proteins, the
Walker A motif (also called P-loop) in the structure of
HisP is heavily involved in coordinating ␤- and ␥-phosphates of ATP. The bound ATP is further stabilized by a
tyrosine residue (Y16) that interacts with the adenine ring
of ATP. The aspartate residue (D178) in the Walker B
motif interacts with a water molecule, which probably
takes the position of the magnesium ion that normally
serves as a cofactor for ATP hydrolysis. In addition, a
glutamate residue (E179) immediately following the
Walker B aspartate and a glutamine residue (Q100) in the
Q-loop region, both highly conserved, form hydrogen
bonds with a water molecule (H2O 437), which interacts
with the ␥-phosphate through a hydrogen bond that is
nearly parallel to the P␥-O␤ bond. Since this P␥-O␤ bond
breaks upon hydrolysis, it is speculated that water 437 is
the most likely candidate for the “attacking” water during
ATP hydrolysis and either Q100 or E179 could be the
activating residue. This structure not only gives us a first
glance of the molecular interactions between ATP and its
binding pocket, importantly, it also provides structural
explanations for many genetic and biochemical data obtained in vivo for the histidine transporter (292). Furthermore, the importance of the glutamate adjacent to the
Walker B motif in ATP hydrolysis is confirmed for numerous other ABC proteins (218, 238, 302, 315), although the
exact chemical mechanism by which this glutamate catalyzes ATP hydrolysis remains controversial (82, 115).
In less than 10 years, the structures of more than 10
other NBDs have been solved at an atomic level (45, 71,
98, 152, 167, 168, 250, 278, 296, 322, 346, 348). Although the
overall sequence homology among NBDs from different
ABC proteins is not very high except in those conserved
motifs, interestingly however, the basic architecture is
remarkably similar even between classical ABC transporters and somewhat distantly related proteins such as
Rad50, a DNA repairing enzyme (130). The prototypical
structure of an NBD consists of an F1-type core subdomain containing the nucleotide binding motifs (Walker A
and B), which is sandwiched by the anti-parallel ␤-sheet
subdomain usually containing an aromatic amino acid
that interacts with the adenine ring of ATP (12), and the
␣-helical subdomain containing the signature sequence
LSGGQ.
B. NBDs Form Dimeric Structures
Although some early reports show that the NBDs are
crystallized as a monomer (152, 346), it is now generally
accepted that there is a dynamic process for two NBDs to
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simply through alterations of the transport pathway to
meet the need of accommodating different substrates
(291). This scene of evolutionary relatedness in all ABC
proteins can be sensed not only from the structure-biological point of view (discussed in sect. VII), but also from
their functional similarities. For example, CFTR is a member of the ABCC subfamily. While CFTR is a genuine ion
channel, carrying out the function of passive diffusion of
negatively charged chloride ions, many related members
of this subfamily, e.g., ABCC1, a leukotriene transporter
(166, 188), actively transport various organic anions.
While it is obligatory for these active transporters to
possess a binding site for their anionic substrates, interestingly, the presence of a binding site for organic anions
in the ion permeation pathway of CFTR has been repeatedly demonstrated (6, 177–179, 206, 280, 289, 321, 354 –
356). Does this binding site for organic anions in CFTR
represent an evolutionary vestige when an active transporter of the ABCC family is converted to an ion channel?
This question will be revisited after we discuss the structure and function of ABC transporters and CFTR.
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TSUNG-YU CHEN AND TZYH-CHANG HWANG
ModBC, from Archaeoglobus fulgidus (129); a metalchelate transporter, HI1470/1, from Haemophilus influenza (234); and a drug exporter, Sav 1866, from
Staphylococcus aureus (65).
NBD dimerization has also been demonstrated with
biochemical assays. Using an analytic gel filtration technique, Moody et al. (218) showed that MJ0796 and MJ1267,
two bacterial ABC transporters’ NBDs, can form a dimer
in the presence of ATP. Neither ADP nor AMP-PNP can
substitute for ATP in the dimerization reaction, suggesting a critical role of the ␥-phosphate in catalyzing dimer
formation. Since the dimer is only seen in mutants whose
ATPase activity is abolished (by mutating the glutamate
residue adjacent to the Walker B aspartate), it is suggested that ATP hydrolysis by wild-type dimers provides
the energy to break the stable dimer formation (218, 296).
Thus, during the normal ATP hydrolysis cycle, the NBD
dimer is short-lived. This speedy dissociation of NBD
dimers is an essential element for an efficient transport
cycle, in which the presence of an absorptive state is not
allowed. Chen et al. (45) showed crystallographically that
MalK, the NBD of bacterial maltose transporters, can
assume two different NBD dimeric structures depending
on whether the structures contain ATP. In the absence of
bound ATP, the structure shows a wide gap (⬎10 Å)
between two NBDs. On the other hand, the gap is closed
in the structure with bound ATP molecules. Based on
these snapshots of NBD dimers, these investigators (45)
proposed a tweezer-like motion upon ATP binding to NBDs
for maltose transporters. This hypothesis is strongly supported by a recent demonstration that the crystal structure
FIG. 4. Cartoon representations of the four full-length structures for ABC transporters. From left to right, structures are BtuCD (pdb 1L7V), a
vitamin B12 importer; SAV1866 (pdb 2HYD), a bacterial drug exporter; HI1470/1 (pdb 2NQ2), a metal-chelate-type ABC transporter; and ModBC (pdb
2ONK), a molybdate transporter, respectively. The two membrane-spanning domains are colored in yellow and magenta. The two nucleotide binding
domains are colored in green and pink, except in SAV1866, where they are colored in their correspondent membrane-spanning domains. Bound
ligands in the nucleotide binding domains are shown in ball-and-stick model and colored by their atom types. No ligand is present in the structure
of HI1470/1. Cyclotetravanadate, ADP, and phosphate are ligands for the NBDs of BtuCD, SAV1866, and ModBC, respectively. It should be noted
that the assembly of two MSDs, as well as the relationship between NBDs and MSDs in Sav1866, is fundamentally different from other structures.
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associate/dissociate (i.e., dimerization). Despite a high
structural conservation among NBD monomers, the first
three crystal structures of NBDs show different dimeric
configurations (71, 130, 135). This dispute had been resolved after more dimeric structures emerged. More recently solved structures of NBD dimers show a head-totail configuration (45, 296, 348). Once an NBD dimer is
formed, the two ATP-binding sites are buried at the dimer
interface. The bound nucleotides as well as many amino
acid residues from each NBD participating in nucleotide
interactions are intimately involved in forming a stable
dimer. Because most of the inter-NBD interactions occur
via the ATP molecules, these are often referred to in the
literature as “glues.” This new dimer structure places the
signature motif (LSGGQ) that defines the ABC transporter
family at the dimer interface involved in interactions with
ATP (especially with the ␥-phosphate of ATP). Thus, once
the NBDs dimerize, the ATP binding pocket is composed
of the Walker A and B motifs from one NBD and the
signature motif from the partner NBD. This assembly of
the ATP binding pocket then satisfactorily explains the
involvement of the signature sequence in ATP binding and
hydrolysis from mutagenesis studies (23, 170, 259, 276,
307, 313) as well as photocleavage and cross-linking studies implicating the close proximity between the signature
sequence and the opposing Walker A motif (93, 191). Most
importantly, this head-to-tail assembly of NBD dimers is
consistent with the dimeric configuration found in the
holoenzyme structures of several bacterial ABC proteins
(Fig. 4), including the vitamin B12 transporter BtuCD
from E. coli(186); a putative molybdate transporter,
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C. Crystal Structures and Working Model
of ABC Proteins
Recent breakthroughs in solving the crystal structures of several ABC transporter holoenzymes (65, 66,
129, 136, 186, 234) not only confirm the long-speculated
domain organization of the ABC proteins, but also allow a
glimpse at the atomic level of the details of individual
domains and their interactions (Fig. 4). Although these
structures show similar, if not identical, arrangement of
the two NBDs, the transmembrane domain folds exhibit a
high degree of structural variability. For importers that
transport its substrate into the cell such as BtuCD,
HI1470/1, and ModBC, not only is the number of transmembrane segments different, the fold of individual MSD
also shows striking diversity. However, one common feature of the importer structures with nucleotide-free NBDs
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is the presence of an inward-facing, central cavity that is
accessible to the cytoplasm (third and fourth structures in
Fig. 4), a likely candidate for the substrate translocation
pathway. In contrast, the structure of BtuCD (first in
Fig. 4) with bound cyclotetravanadate in its NBDs contains an outward-facing, central pathway. It is interesting that these pathways are only access to either the
cytoplasm or the external medium as if they are “gated”
on one end or the other by the respective transmembrane segments.
Some surprising findings emerge from the crystal
structure of an exporter, Sav 1866 (second structure in
Fig. 4). Compared with the domain arrangement seen in
all the importers shown in Figure 4, the relationship between MSDs and NBDs in Sav 1866 takes a new twist.
While the NBD dimeric structure of Sav 1866 is conserved, each NBD interacts not only with the corresponding MSD, but also with the MSD of the partner subunit.
Thus each MSD straddles on both NBDs via its two intracellular loops (Fig. 4). This arrangement between MSDs
and NBDs makes it difficult to envision a simple translational movement of the two subunits of an ABC transporter and necessitates a twisting motion during a transport cycle (65). Equally surprising is the observation that
the NBD dimer interface of Sav 1866 with two bound ADP
molecules (therefore suggesting a posthydrolytic conformation) is extremely similar to that of hydrolysis-defective MJ0796 with bound ATP, suggesting a limited separation of two NBDs during the cycle of ATP binding/
hydrolysis as proposed by Jones and George (150).
However, it is lately proposed that this ADP-bound structure of Sav 1866 actually represents a prehydrolytic ATPbound conformation, since a similar overall structure of
Sav 1866 was observed when the NBDs are occupied by
the nonhydrolytic ATP analog AMP-PNP (66). This latter
scenario assumes that AMP-PNP binding to NBDs can
trigger NBD dimerization, although, at least for isolated
NBDs of MJ0796 and Md11P, nonhydrolyzable ATP analogs fail to dimerize NBDs (141, 218). Nevertheless, the
presence of an outward-facing pathway in Sav 1866
prompts Locher and colleagues (128) to propose a common mechanism of coupling ATP binding/hydrolysis to an
alternating substrate binding site for both importers and
exporters. In their model, MSDs flip from the inwardfacing to the outward-facing configuration following NBD
dimerization upon ATP binding. ATP hydrolysis and dissociation of ADP and phosphate reset the system to the
original conformation. This model of an alternating substrate binding site for ABC transporters bears remarkable
resemblance to the one proposed for the lactose permease (109) and the “rocking banana” cartoon depicted in
Figure 1. The difference between an importer and an
exporter is that while an importer translocates the substrate by ATP binding and subsequent NBD dimerization,
the translocation of the substrate across MSDs of an
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of MalK in the posthydrolysis state shows a disengagement of ADP-bound Walker A and B motifs from the
signature sequence of the partner NBD (192). The numerous structural and biochemical studies described above
have generated a popular hypothesis that ATP bindingtriggered NBD dimerization may serve as the power
stroke to do mechanical work; hydrolysis of the bound
ATP drives the dissociation of the NBD dimer, and subsequent release of the hydrolytic products, ADP and Pi,
completes the transport cycle (125, 219).
By comparing NBD structures with or without ligands and structures in the presence of different nucleotides, one can learn about possible conformational
changes induced by ATP binding and hydrolysis. Karpowich et al. (152) reported that ATP binding to the
Walker A motif likely induces conformational changes
involving the conserved D-, Q-, and H-loops, regions participating in interactions with the ␥-phosphate of ATP. It
is suggested that this induced-fit mechanism probably
also plays a role in facilitating NBD dimer formation.
The same report also showed an outward rotation of
the LSGGQ-containing ␣-helical subdomain of the NBD in
ADP-bound monomeric structure of MJ0796. Since the
␣-helical domain interacts directly with MSDs of ABC
transporters (see sect. VIIC), if we accept the idea that this
ADP-bound monomer represents a posthydrolysis structure of NBD, this inward movement by ATP binding and
outward motion following ATP hydrolysis of the ␣-helical
subdomain may well be part of the signaling transmission
mechanism relating NBD machine and the transport pathway in MSDs. While these structural/biochemical studies
of isolated NBDs have provided important mechanistic
insights into the molecular motion of NBDs upon ATP
binding/hydrolysis, one apparent caveat of these approaches is the absence of MSDs, which could affect the
conformational freedom of NBDs.
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hydrolysis at one ATP binding pocket has to be able to
sustain the transport function for members in the ABCC
subfamily. Perhaps, the presence of two ATP binding
sites simply improves the kinetics of substrate transport if ATP binding to one NBD could initiate the
transport process. If the primordial ABC transporters
possess two identical MSDs and NBDs, and other asymmetrical ABC transporters evolved through gene duplications and mutations, one can speculate that the NBD
association/dissociation process of all ABC proteins
during the transport cycle may require only a single
hydrolysis-competent site (315). This idea is consistent
with a recent biochemical study on the NBDs of hemolysin B translocase (349). By mixing wild-type and hydrolysis-deficient mutant NBDs, Zaitseva et al. (349)
showed that one single hydrolysis-competent site may
be sufficient for completion of an ATP-dependent
dimerization cycle (cf. Refs. 131, 141). A modified but
similar approach was undertaken for MJ0796. When
mixing two hydrolysis-deficient NBDs, one with the
signature sequence serine mutated and the other with
the Walker B glutamate mutated, ATPase activity is
recovered, again suggesting that a single active site is
sufficient to complete the reaction cycle in the soluble
cassettes (P. Thomas and J. Moody, personal communication).
There is little doubt that recent crystallographic studies of ABC transporter proteins have offered unprecedented insight into how these transporters work, yet we
should bear in mind that these structures very often were
solved under nonphysiological conditions. The symmetrical structures of NBD dimer with two ATP molecules
sandwiched in between each NBD monomer may not
necessarily represent a native state (see Ref. 150 for
extensive discussion). It is known that substrate binding
can affect the NBD function in ABC proteins (162, 181; cf.
Refs. 201, 202). It is possible that a stochastic binding of
one substrate molecule to one site activates the physically
coupled NBD; subsequent ATP binding to this particular
NBD triggers NBD dimerization which will exclude ATP
binding to the other NBD. Alternatively, at the end of an
ATP hydrolysis cycle by one NBD, retaining the hydrolytic
product, e.g., ADP, can prevent ATP binding to this particular NBD. Therefore, there are multiple ways to make
two symmetrical ATP binding sites function asymmetrically (162). Molecular dynamic simulations also show that
docking and subsequent occlusion of ATP at one catalytic
site of BtuCD is coupled to loosening of the nucleotide
binding pocket at the second site (230; cf. Ref. 229).
Furthermore, in a P-gp mutant where both catalytic glutamate residues are mutated to alanine, ATP was found
occluded in just one site (315), consistent with earlier
reports that in wild-type P-gp, one active site is empty
when the other site is occupied by the vanadate-ADP
complex (248, 319). More kinetic studies using crystal
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exporter is accomplished by ATP hydrolysis and dissociation of the hydrolytic products.
Close inspections of the interface between NBDs and
MSDs also reveal intriguing structural features that may
have functional implications. In all the structures shown
in Figure 4, one can observe a short ␣-helix connecting
NBDs and MSDs. In these four different ABC transporters,
although these helices come from different intracellular
loops of the MSDs, they are all embedded in a groove
between the core and helical subdomains of NBD, where
they interact extensively with the amino acids around the
conserved Q-loop. Because of the physical location of this
helix, it is considered the “coupling helix” (128) that
transmits the molecular events between NBDs to MSDs. It
is worth noting that while each MSD of an importer
interacts with the NBD via one single coupling helix, the
structure of Sav 1866 suggests the presence of two coupling helices per half molecule: one interacts with both
NBDs and the other with the NBD of the opposite halfmolecule. The functional importance of these intracellular loops is demonstrated by the reports that gating defects are observed for disease-associated mutations in all
four intracellular loops of CFTR (58, 285–287).
Exactly how to relate the chemical reactions at the
two ATP binding sites to the kinetic steps of a transport
cycle remains unclear and controversial, although for
most ABC transporters, both ATP binding sites are capable of hydrolyzing ATP (46, 226, 306, 318). Although it is
technically challenging to accurately estimate the ATP to
substrate stoichiometry, one report suggests two ATP
molecules hydrolyzed for one molecule of substrate transported (231). Since it is unlikely that two ATP molecules
are hydrolyzed simultaneously, each hydrolysis event is
probably coupled to a distinct step of one transport cycle
as reported for P-glycoproteins (P-gp) (272, 273). This
hypothesis then suggests that mutations that abolish one
hydrolysis cycle will be sufficient to halt the entire transport cycle. However, for HisP, when one of the two ATP
binding sites is mutated to abolish its ATP hydrolysis
capability, the resulting mutant can still transport histidine, although at half the rate of wild-type proteins (226).
In addition, mutations of the catalytic glutamate at one of
the NBDs in P-gp only partially inhibit both basal and
drug-stimulated ATPase activity (314). Interestingly, the
magnitude of inhibition differs in these two partially hydrolysis-deficient mutants. Thus one also needs to entertain the alternative scenario that ATP hydrolysis occurs
alternately and one ATP hydrolysis cycle is sufficient to
complete the transport cycle of the substrate (288). Although the exact stoichiometry between the ATP hydrolysis cycle and the transport cycle remains debatable for
ABC transporters with two hydrolysis-competent sites, in
many of the ABC proteins (notably in the ABCC subfamily) including CFTR, the NH2-terminal NBD lacks the catalytic glutamate adjacent to the Walker B motif. Thus
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CLC-0 AND CFTR
structures as a guide are needed to elucidate the sequence
of molecular events during a transport cycle.
VIII. CFTR CHLORIDE CHANNEL
A. Introduction
Physiol Rev • VOL
FIG. 5. Simplified topological model emphasizing the postulated
domain structure of a CFTR chloride channel, including cytoplasmic
amino (N) and carboxy (C) termini, two nucleotide binding domains
(NBD1, NBD2), a regulatory (R) domain, and the predicted membranespanning ␣-helices (TM1–TM12). Each NBD contains one ATP-binding
site. The ATP binding site is characterized structurally by the Walker A
lysine (K464 or K1250), which coordinates ␤- and ␥-phosphates of the
bound ATP, Walker B aspartate (D571 or D1370), which coordinates
Mg2⫹, and the catalytic glutamate (E1371, but S573 in NBD1). The
signature sequence of NBD2, together with the Walker A and B motifs of
NBD1, forms the second ATP binding pocket (ABP1) in an NBD dimer.
Similarly, the ABP2 is formed by the signature sequence of NBD1 and
the Walker A and B motifs of NBD2 (see Fig. 6B).
of many of the consensus serine residues in the R domain
is a prerequisite for CFTR to function. After the channel is
phosphorylated by protein kinase A (PKA) and ATP,
opening and closing (i.e., gating) of phosphorylated channels is controlled by ATP. Numerous mutagenesis studies
have indicated that phosphorylation affects CFTR activity
in a very complicated manner. Not only is the activity of
CFTR incrementally regulated by differential phosphorylation of the PKA consensus sites, protein kinase C-dependent phosphorylation may also modulate CFTR. Interested readers are referred to several recent articles that
have extensively reviewed this subject (96, 117, 284, 290,
358). Here we focus on ATP-dependent gating, which is
likely to have an evolutionary relationship to the transport
function for other ABC proteins.
B. Crystal Structures of CFTR’s NBD1
Before we discuss CFTR gating function and its
mechanism, we first review recent advances in solving the
crystal structure of human and mouse CFTR’s NBD1 by
Lewis et al. (167, 168) and Thibodeau et al. (310; also see
Ref. 250 for the NBD structure of another member of the
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CFTR (ABCC7; Ref. 264) is one of the very few
members of the ABC proteins that does not function as an
active transporter (one other analogous member is sulfonylurea receptor, SUR or ABCC8). Unlike SUR, which
acts as a regulatory subunit for KATP channels (reviewed
in Ref. 224), CFTR by itself functions as a chloride channel as demonstrated by Bear et al. (26) with purified CFTR
in reconstituted lipid bilayers. Mutations that diminish the
CFTR channel activity cause cystic fibrosis (CF), the most
common, lethal genetic disease in Caucasians that affects
1 in ⬃2,500 newborns in the United States (334). In CF
patients, normally Cl⫺-permeant epithelial cells in airways, pancreas, and other tissues become Cl⫺ impermeable, causing defective salt, water, and protein transport.
On the other hand, increased activity of the CFTR Cl⫺
channel, usually caused by bacterial toxins, results in
secretory diarrhea that incapacitates millions of people
each year in developing countries (30). Therefore, understanding how the CFTR channel is gated will have a direct
impact on the development of novel therapeutics for
treatment of CF and secretory diarrhea.
As a member of the ABC superfamily, CFTR contains
the characteristic architecture of two MSDs and two
NBDs (NBD1 and NBD2) (Fig. 5). There is strong evidence that CFTR’s two NBDs form a head-to-tail dimer
similar to those found in other ABC transporters (323).
The two ATP binding pockets (ABP) for CFTR are defined
as follows: ABP1, formed by the Walker A and B motifs of
NBD1 and the signature sequence of NBD2; ABP2, formed
by the Walker A and B motifs of NBD2 and the signature
sequence of NBD1. Similar to other members of the ABCC
subfamily, the amino acid sequences of CFTR’s two NBDs
show significant differences even in those conserved motifs. For example, the glutamate residue adjacent to the
Walker B motif is replaced by a serine residue in NBD1. A
histidine residue that has been shown to play an important role in ATP hydrolysis in other ABC proteins (e.g.,
HlyB) (348) is also replaced by a serine in NBD1. In
addition, the signature sequence in CFTR’s NBD2 is somewhat degenerate (LSHGH instead of LSGGQ). This structural asymmetry of the two NBDs in CFTR likely accounts
for the observation that only ABP2, but not ABP1, hydrolyzes ATP at an appreciable rate (9, 24, 302).
In addition to the canonical MSDs and NBDs seen
with other ABC proteins, CFTR contains a unique regulatory (R) domain located between the NH2-terminal NBD
(or NBD1) and the second MSD (Fig. 5). Phosphorylation
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TSUNG-YU CHEN AND TZYH-CHANG HWANG
Physiol Rev • VOL
S660 and S670 in the regulatory extension). However,
only S660 and S670, but not S422, have been shown to be
phosphorylated in vitro or in vivo (96). While the structural details of the 30-amino acid insertion is not completely solved in the crystal structure, implicating flexibility of that part of the molecule, the physical location of
the regulatory extension may impart functional implications. According to sequence analysis, the regulatory extension, an ␣-helix in the crystal structure, belongs to the
R domain. This helix, if present at the same location of the
intact CFTR protein, would hinder NBD1/NBD2 dimer
formation because of its location at the presumed dimer
interface. Indeed, fitting NBD1 and modeled NBD2 into
the MJ0796 dimer results in significant steric clashes (167,
220; Zou and Hwang, unpublished observations). This
raises an interesting possibility that the role of this region
together with other parts of the R domain is to prevent
dimer formation; phosphorylation of the R domain may
result in a conformation change that enables NBDs to
dimerize. It should be noted that removal of this segment
does not result in PKA-independent activity, indicating
that this helix is not required for preventing dimer formation in nonphosphorylated CFTR (59). On the other hand,
removing a larger part, or all, of the R domain renders the
channel activity phosphorylation independent (33, 60,
261). Recent studies using cross-linking strategies for assessing NBD dimerization in CFTR also showed that PKA
phosphorylation indeed promotes dimerization of CFTR’s
two NBDs (207). However, it is important to note that the
functional role of the R domain is more than a simple
inhibition of CFTR opening (see details below).
While these NBD1 structures of CFTR have served as
a guide for functional studies of the roles of the NBDs in
channel gating (323, 360), the field still awaits high-resolution structures of the whole CFTR protein. Because of
the technical difficulties in obtaining a large quantity of
purified CFTR proteins, crystallization of CFTR remains a
mounting challenge. Nevertheless, Ford and colleagues
(21, 265) have made the first leap in solving the lowresolution (⬃20 Å) structures of CFTR from two-dimensional crystals. The overall shape and size are similar to
those of the low-resolution structure of P-gp solved earlier (266). Since the two-dimensional crystal of CFTR was
grown in the presence of AMP-PNP, a nonhydrolyzable
ATP analog that can keep the channel in the open state
(113, 138), it is inferred that one of the observed structures represents an open-channel conformation. It should
be noted, however, that AMP-PNP by itself is a poor
ligand to open CFTR channels (324; cf. Ref. 7). In addition, even the hydrolysis-deficient mutant E1371S-CFTR
seldom assumes a stable open state in the presence of
AMP-PNP alone (Cho and Hwang, unpublished data).
These data underscore the importance of the exact configuration of the ␥-phosphate in determining the structure
of NBD dimers (150). Therefore, more studies are needed
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ABCC subfamily). Not surprisingly, the basic architecture
of these two solved NBD1s is very similar to known NBD
structures. All three characteristic subdomains described
above are conserved. Also as seen in NBDs from other
members of the ABC proteins, the bound MgATP is coordinated by the Walker A motif (K464 and S465 in human
CFTR) and the Walker B aspartate (D572). However, as
described above, two critical residues (Walker B glutamate and histidine in the “switch” region) that form hydrogen bonds directly or indirectly with the ␥-phosphate
in NBDs of other ABC proteins (135, 296) are replaced by
serine. This likely explains why biochemical studies demonstrate that ATP is hydrolyzed in ABP2 but not in ABP1
(9, 24, 302). This structural/biochemical asymmetry, as
described above, is an interesting feature of the ABCC
subfamily (as well as many ABC transporters in bacteria, yeasts and fungi, see Ref. 151). The functional
implications of this structural asymmetry will be discussed below.
Another interesting feature revealed by the structure
of mouse CFTR’s NBD1 is a nonclassical interaction between ATP and the binding pocket. Instead of a trans
configuration for the backbone ␥ torsion angle between
the ribose and the nucleotide base usually seen in other
ABC proteins (e.g., Ref. 296), the ATP molecule in mouse
CFTR’s NBD1 assumes an unusual gauche ⫹ configuration. Unlike a classical ring-ring stacking interaction between ATP and an aromatic amino acid (e.g., tyrosine-11
in the previously mentioned crystal structure of MJ0796),
the nucleotide base in the structure of mouse NBD1 (167)
seems to be gripped by three hydrophobic amino acids
(W401, L409, and F430). However, in the structure of
modified human NBD1 (168), a canonical stacking interaction between the nucleotide base and the W401 residue
is observed. Although the reason for this discrepancy
remains unclear, it should be noted that each crystal
structure only reflects a snapshot of the protein. Furthermore, some of these amino acids (e.g., F409 and F430)
that interact with the nucleotide base are located in a
region that may be structurally dynamic (as implicated by
the elevated B-factors in the crystallographic data set). It
is possible that these two structures of NBD1 represent
NBD1 conformations appropriate for different states of
the channel or the mutations made in human NBD1 to
facilitate crystallization could potentially change the
structure. Alternatively, the unusual ATP-W401 interaction in the first CFTR NBD1 crystal is caused by crystal
lattice interactions that influence side-chain rotamers and
configurations (310).
Also unique to CFTR’s NBD1 is the presence of a
regulatory insertion region (amino acids 404 – 435) at the
NH2 terminus and a regulatory extension (amino acids
639 – 670) at the COOH terminus. It is interesting that both
regions contain consensus serines for PKA-dependent
phosphorylation (S422 in the regulatory insertion and
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CLC-0 AND CFTR
to ascertain the functional state of those low-resolution
structures.
C. CFTR Function
Physiol Rev • VOL
D. Roles of ATP Hydrolysis in CFTR Gating
The involvement of ATP hydrolysis in CFTR gating
was implicated even in the earliest studies. Anderson
et al. (15) and Nagel et al. (221) reported that ATP, not
AMP-PNP or ATP␥S (at 0. 5–1 mM), can open PKA phosphorylated CFTR. This observation was taken as evidence
for an involvement of ATP hydrolysis in opening the
phosphorylated CFTR. Further support for this idea was
the observation that Mg2⫹, a cofactor for ATP hydrolysis,
is required for normal channel opening by ATP (15, 72,
170). Since AMP-PNP, when applied together with ATP,
locks the channel in a stable open state that can last for
minutes, it was proposed that ATP hydrolysis also controls channel closing (42, 113, 138). This AMP-PNP-induced, locked-open state is simulated by mutating the
Walker A lysine at NBD2 (112), an amino acid residue that
is critical for ATP hydrolysis (251). Therefore, it was
proposed that hydrolysis by NBD2 closes the channel.
Furthermore, since mutating the equivalent lysine at
NBD1 (i.e., K464) decreases the opening rate (42, 112; cf.
Refs. 237, 251), it was hypothesized that ATP hydrolysis at
NBD1 controls channel opening. Thus two hydrolysis
events at CFTR’s NBD1 and NBD2 were thought to be
distinctly coupled to opening and closing of the channel
(72, 350).
While this once popular model for CFTR gating can
explain many early functional data, as more results
emerged, however, its validity became questionable. Aleksandrov et al. (8) first demonstrated that 5 mM AMP-PNP
or ATP␥S can open phosphorylated CFTR in lipid bilayers. This observation was later confirmed in excised, inside-out patches by Vergani et al. (324), although the
results with AMP-PNP in these two studies are quite
different quantitatively. These studies cast serious doubt
on the early notion that ATP hydrolysis is obligatory for
channel opening (112, 138). It could well be that AMPPNP is simply a poor agonist, an idea supported by many
structural/biochemical studies of ABC proteins described
in section VIIB, but sequence analysis and structural studies (167, 168) described above also suggest that CFTR’s
NBD1 lacks the essential catalytic base for ATP hydrolysis. Moreover, photolabeling experiments with 8-azidoATP, a hydrolyzable ATP analog that can sustain CFTR
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Functional properties of CFTR have been extensively
investigated ever since the gene was cloned in 1989 (264).
Since the goal of the current review is to explore the
structural/functional relationship between transporters
and ion channels, we will confine our discussion to ATPdependent gating, which is likely to be the analogous
behavior of the transport function for other ABC proteins.
The permeation properties of the CFTR pore and the cell
biological aspects of CFTR function are discussed at
length in Dawson et al. (64) and Guggino and Stanton
(111), respectively. The technical aspects of gating studies of CFTR chloride channels, covered by other reviews
(236, 358), are not repeated in the current article.
Several years before the CFTR gene was cloned, Paul
Quinton’s elegant electrophysiological work on isolated
sweat ducts has implicated a defective chloride conductive pathway as the fundamental pathophysiological
mechanism for CF (249; cf. Ref. 154). It was found that the
transepithelial potential of sweat ducts isolated from CF
patients is significantly more negative than that of normal
controls. This abnormal bioelectric potential in CF sweat
ducts can be simulated in normal control ducts simply by
replacing chloride ions with impermeant anions, indicating an involvement of a chloride permeation pathway that
is defective in CF. This defective chloride conductive
pathway then accounts for the elevated sweat [chloride],
a diagnostic hallmark in CF. When the CFTR gene was
cloned in 1989, it was somewhat puzzling initially why
the CFTR protein shows a characteristic topology of an
active transporter (264). This mystery was quickly
solved by the demonstration that, in excised, inside-out
membrane patches, hydrolyzable nucleoside triphosphates, such as ATP, GTP, ITP, CTP, and UTP, are
required to activate CFTR chloride currents following
PKA-dependent phosphorylation in a heterologous expression system that does not express native CFTR
(15). Since single-channel chloride currents can be
readily discerned, the most likely explanation for these
observations is that CFTR itself encodes a chloride
channel whose function is controlled by PKA-dependent phosphorylation and the presence of ATP. Several
other studies solidify this conclusion. First, Anderson
et al. (16) demonstrated that mutating amino acid residues in the putative pore-forming domain of CFTR
alters the anion selectivity. Second, Rich et al. (261)
show that partial removal of the R domain renders the
CFTR channel phosphorylation independent (261).
Lastly and most importantly, Bear et al. (26) painstakingly purified CFTR and incorporated the protein into
reconstituted lipid bilayers. A chloride channel with all
the characteristic biophysical properties as the channels found in native epithelia (105) or cells heterologously expressing CFTR was observed. This latter
study leaves no doubt that CFTR itself is a chloride
channel. It should be noted, however, the fact that
CFTR itself is a chloride channel does not rule out the
possibility that CFTR may carry out other functions
(158, 283).
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TSUNG-YU CHEN AND TZYH-CHANG HWANG
gating effectively, consistently show that CFTR’s ABP1
does not hydrolyze ATP at an appreciable rate (9, 24, 305;
cf. Refs. 153, 155). These results essentially disprove the
idea that ATP hydrolysis at ABP1 controls channel opening. However, since CFTR mutants whose ATP hydrolysis is
abolished (e.g., K1250A, E1371S) (251, 302), once opened by
ATP, can remain open for minutes (34, 112, 323, 324, 350;
cf. Refs. 42, 251), it is now generally accepted that hydrolysis of ATP at ABP2 closes the channel (97, 358). Thus,
unlike classical ligand-gated ion channels, CFTR’s ligand
ATP is “consumed” during each gating cycle.
As discussed in section VII, the two NBDs of the ABC
proteins form a head-to-tail dimer upon ATP binding. In
most cases, this dimer formation is observed when ATP
hydrolysis is abolished by mutations (296). It is thus speculated that for the wild-type NBDs, ATP hydrolysis causes
fast separation of the two NBDs. This rapid dissociation of
NBDs ensures an effective transport cycle. That CFTR’s two
NBDs may assume a prototypical head-to-tail configuration
was elegantly demonstrated by Vergani et al. (323). Through
statistical analysis of more than 20,000 NBD sequences,
they identified a pair of amino acids (one, R555, in NBD1
and the other, T1246, in NBD2) that likely coevolved as a
pair to maintain the ability of forming a hydrogen bond at
the dimer interface. Indeed, in several known structures
of NBD dimers, these two amino acids are rightfully oriented to form a hydrogen bond (45, 296, 349). After carrying out a series of well-designed mutant cycle analyses,
Vergani et al. (323) made an insightful conclusion that
these two amino acids (R555 in the signature sequence of
NBD1 and T1246 in the Walker A motif of NBD2) do not
interact when the channel is in the unliganded or liganded
closed state, but they do interact when the channel is
open or in the transition state. Thus one can envisage that
NBD1 and NBD2 undergo a dynamic association/dissociation that is coupled to opening/closing of the channel. It
is interesting to note that the functional importance of
this hydrogen bond pair in CFTR is attested by the fact
that mutations of either amino acid are associated with
CF (CF Consortium, http://www.genet.sickkids.on.ca/
cftr/). More recently, Mense et al. (207) engineered a
series of cysteine pairs (one at NBD1 and the other at
NBD2) based on the modeled NBD dimer structure of
CFTR. Cross-linking experiments with sulfhydryl reagents
not only confirmed that CFTR’s two NBDs form a dimer
with a conserved interface, but also provided evidence
supporting the notion that PKA phosphorylation promotes dimer formation. Surprisingly, however, when two
engineered cysteines at the dimer interface form a disulfide bond, the channels still show slow open-close transiPhysiol Rev • VOL
F. Are the Two ATP-Binding Sites
Functionally Distinct?
Although it is now well accepted that each of the two
ATP binding pockets in an ABC protein consists of components from both NBDs, it should be noted, however, in
all the crystal structures of monomeric NBDs (including
CFTR’s NBD1), that ATP is bound to the Walker A and B
motifs, but not the signature motif, suggesting that the
signature sequence may not contribute to ATP binding
before two NBDs are dimerized. Nevertheless, in theory,
to understand the functional role of ATP binding at each
site, one can manipulate the Walker motifs and/or the
signature sequence. Many mutagenesis studies have been
undertaken along this direction.
Before we cover these studies, a few technical issues
that can affect how electrophysiological data are interpreted should be discussed. Single CFTR channel currents show characteristic long bursts of opening (Fig. 6A).
Each burst, lasting for hundreds of milliseconds to seconds, is interrupted by brief flickery closings with a time
constant of ⬃10 ms. Thus the closed-time histogram can
be at least fitted with two components (113), with the
longer closed-time constant being a function of [ATP] (34,
339). The short flickery closing within a burst is voltage
dependent, but independent of [ATP]. These events are
likely due to blockade of the CFTR pore by yet-to-beidentified cytoplasmic anion blockers (308, 357; cf. Ref.
40). Different methods have been employed to isolate the
ATP-dependent events during single-channel kinetic analysis (7, 34, 60, 339). Thus kinetic parameters reported
from different groups vary tremendously even for wildtype CFTR. For hydrolysis-deficient mutants such as
K1250A, the bursting time estimated with different methods in different reports can differ by ⬃100-fold. For example, using macroscopic current relaxation analysis,
Zeltwanger et al. (350) and Powe et al. (237) reported a
locked open-time constant of ⬃2–3 min for K1250ACFTR. However, a bursting duration of ⬃1 s was estimated for the same mutant with microscopic kinetic analysis (42, 251). Different expression systems used by different groups may further complicate the issue (see a
summary in Ref. 358). It is thus very difficult, if not
impossible, for the authors to make a comprehensive
comparison among all the results in the literature. Instead, with a few exceptions, we set our priority in discussing papers that apply relatively similar kinetic analy-
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E. Coupling of NBD Dimerization
to Gating Transitions
tions, suggesting that the coupling of NBD dimerization
and opening/closing of the gate may be more complicated.
For example, Csanady et al. (61), by examining temperature dependence of CFTR gating, reported evidence that
the NBD dimer is already formed but the gate (presumably located in MSDs) is yet to open.
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CLC-0 AND CFTR
sis methods as ours. In light of these complications, we
apologize in advance if certain papers are not well presented.
Since there are two ATP-binding pockets (ABP1 and
ABP2) in CFTR, questions relating these two binding sites
to CFTR gating include the following: 1) Which ATP
binding site is coupled to the ATP-dependent opening?
2) Is binding of ATP to both sites necessary to open the
channel? 3) Since only ABP2 contains the catalytic glutamate for ATP hydrolysis, what is the role of ATP binding
at ABP1 in CFTR gating? 4) What is the functional role of
the signature sequence? This last question is especially
important since many CF-associated mutations are found
in the signature sequence of both NBDs.
As described above, several early studies suggest a
role of ATP binding at ABP1 in channel opening based on
studies of mutations at the Walker A lysine residues, K464
and K1250 (42, 112, 350). This idea was first challenged by
Ramjeesingh et al. (251). They showed that purified
K464A-CFTR exhibits a nearly identical opening rate as
wild-type channels at 1 mM ATP. Powe et al. (237) not
only confirmed this result, but also showed that the relationship between [ATP] and the opening rate is not afPhysiol Rev • VOL
fected by the K464A mutation, thus casting serious doubt
on the role of ATP binding at ABP1 in channel opening.
However, two other studies provide evidence for
the essential role of ATP binding at both ABPs for
channel opening (29, 324). Vergani et al. (324) reached
this conclusion based on the observation that mutations of Walker A lysine (K464 and K1250) or Walker B
aspartate in NBD2 (D1370) decrease the apparent affinity for ATP (cf. Ref. 17). Single-channel kinetic analysis suggested that it is the relationship between the
opening rate and [ATP] that is affected by these mutations (324; cf. Ref. 237). It seems safe to assume that
these mutations affect ATP-binding affinity, since crystal structures of the NBD-ATP complex in many ABC
proteins indicate that these charged residues interact
with MgATP. It is therefore concluded that ATP binding
at either site can be made rate-limiting for channel
opening (97). This then leads to the suggestion that
channel opening takes place after both ABPs are occupied by ATP. Berger et al. (29) tackled the same issue
with a different approach. They showed that the opening rate is diminished by introducing bulky entities into
either ATP binding pockets. Since photolabeling with
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FIG. 6. A: single-channel current trace of WT-CFTR. CFTR channels open in bursts interrupted by closed events that last for hundreds of
milliseconds to a few seconds. These long-closed events are [ATP] dependent. As the [ATP] is decreased, the time constant for these closed events
increases. However, even at 2.75 mM ATP, a saturating [ATP], the mean closed time for these closed events is still ⬃400 ms. This is a unique feature
for CFTR as a ligand-gated channel. For classical ligand-gated channels, the ligand-bound closed state has an opening rate that is more than
1,000-fold higher than that of ATP-bound CFTR. Thus for CFTR channel opening, there exists a very slow rate-limiting step following ligand binding
(perhaps reflecting a large conformational change associated with opening of the gate). The expanded trace shows one closed-open-closed gating
cycle. Within one single opening, flickery closings (*) with a time constant ⱕ10 ms can be seen. These events are voltage dependent but ATP
independent. B: a simplified kinetic model for CFTR gating proposed by Zhou and Hwang (358). The two NBDs dimerize upon ATP binding in a
head-to-tail configuration. The head of each NBD, containing the Walker A and Walker B motifs, is represented by a lighter color, while the tail,
containing the signature sequence, is represented by a darker color. In the absence of ATP, the channel can open, but this spontaneous
ATP-independent opening rate is fairly small, ⬃0.006 s⫺1 in Bompadre et al. (33, 35). When the ABP1 site is occupied by ATP (C2 state), the opening
rate is not increased. However, binding of ATP to ABP2 increases the opening rate irrespectively if ABP1 is occupied or not. Once the channel is
opened by ATP binding to ABP2, hydrolysis of ATP rapidly closes the channel (⬃ 2–3 s⫺1). When ATP hydrolysis is abolished (e.g., by mutations),
the nonhydrolytic closing proceeds at a slow rate (⬍0. 01 s⫺1). It is interesting to note that the spontaneous ATP independent open state (O1) is
unstable-time constant ⬃400 ms in Bompadre and co-workers (33, 35), although this closing does not involve ATP hydrolysis. For simplification, the
ATP-independent opening event is depicted as dimerized NBDs, but there is so far no evidence for this proposition. In fact, ATP-independent,
spontaneous openings are observed in a CFTR mutant whose NBD2 is completed deleted (329). Since the spontaneous openings for the
hydrolysis-deficient mutant E1371S are short-lived events (Cho and Hwang, unpublished observations), a stable NBD dimer formation requires the
action of ATP presumably at ABP2. ATP binding at the ABP1 may also contribute to the stability of the open state (NBD dimer) (34, 237, 360). A
restrained version of CFTR gating model can be seen in Vergani et al. (324).
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tant for NBD dimerization (323) is only present in ABP2,
but not in ABP1. In addition, whether ATP binding at
ABP1 is essential for channel opening by ATP binding at
ABP2 is questioned since, unlike the Y1219G mutation,
the W401G mutation in the ABP1 has little effect on the
apparent affinity for ATP in both macroscopic and microscopic measurements (360).
Although the mutations that presumably decrease
ATP affinity at the ABP1 (K464A and W401G) have questionable effects on the ability of ATP to increase the
opening rate of CFTR, both mutants show a shortened
open time, suggesting a destabilization of the open state
by the mutations (237, 360). The effect of K464A on the
open time was seen even in early studies (42, 251). The
shortened opening bursts of K464A-CFTR, compared with
wild-type channels, are visually discernable in Ramjeesingh et al. (251) and Powe et al. (237). In contrast, Vergani
et al. (324) reported that the open time for K464A-CFTR is
not significantly different from that of wild-type channels.
Although it is unclear why the same mutation behaves
differently in different laboratories, differences in the expression system may be partly responsible. It is, however,
important to note that the K464A mutation does shorten
the locked open time of hydrolysis-deficient mutants in
two different studies (237, 324), suggesting that this effect
on the open time is not due to a potential allosteric action
of the mutations at the ABP1 on the ATP hydrolysis rate
at the ABP2, which normally determines the rate of channel closing.
Based on the idea that CFTR’s open channel conformation is associated with an NBD dimer embedded with
two ATP molecules (323), these results can be explained
by an energetic mechanism since the ligand-binding energy will be part of the overall energetics of the openchannel conformation. Thus a reduction of the free energy of ATP binding could be reported as a decreased
open-time constant as seen with the K464A and W401G
mutants. If we consider ATP as a “glue” that bonds two
NBDs together, mutations that loosen the strength of the
interaction between the glue and NBDs could destabilize
the NBD dimer. This mechanism also predicts that an ATP
analog with a higher binding affinity (i.e., a “superglue”)
should stabilize the open state. Zhou et al. (359) demonstrated that N6-(2-phenylethyl)-ATP (or P-ATP), an N6modified ATP analog that has been shown to bind more
tightly to other ATP-binding proteins than ATP (e.g., Ref.
103), activates CFTR with a 50-fold higher apparent affinity than ATP. P-ATP indeed prolongs the open time of
WT-CFTR. P-ATP also increases the locked open time of
the hydrolysis-deficient mutant E1371S-CFTR, suggesting
that this effect of P-ATP is through a tighter binding. The
observation that P-ATP also prolongs the locked open
time of WT-CFTR with AMP-PNP is particularly interesting (359). If the conventional idea is correct that when
CFTR is locked opened by the combined action of ATP
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8-N3-ATP to either ABP is also decreased by these
maneuvers, they concluded that ATP binding to both
ABPs is required to open the channel. However, it is
unclear if the decrease of the opening rate is due to a
decrease of ATP binding affinity rather than due to a
steric hindrance for NBD dimerization resulting from
the introduced bulky side chains. It will be interesting
to test if increasing [ATP] can restore the normal opening rate since an effect on ATP binding affinity is expected to be amended by increasing [ATP].
A major disadvantage with mutating conserved
Walker A and Walker B residues, particularly at the NBD2
site, is that these mutations likely affect ATP hydrolysis.
For example, the hydrolysis-deficient mutant K1250ACFTR, once opened by ATP, stays open for minutes (but
cf. Refs. 42, 251). This prolonged open time exerts tremendous effects on the ATP dose-response based on
macroscopic current measurement, an assay that is usually used as the first step to assess mutational effects on
ATP sensitivity. To circumvent this problem, Zhou et al.
(360) initiated a new approach. Instead of examining the
residues that interact with the phosphate group of ATP,
they focused on aromatic amino acid residues that coordinate the adenine ring of ATP (12). In the modified
human NBD1 structure, an aromatic residue, W401, interacts directly with the adenine ring of ATP by a ring-ring
stacking mechanism (168). A homology model of CFTR’s
NBD2 based on the human CFTR’s NBD1 structure was
made. Together with sequence analysis, a conserved residue Y1219 in NBD2 (thus in ABP2) was identified as the
counterpart of W401 in NBD1 (or ABP1).
When Y1219 is mutated to a glycine (Y1219G), the
ATP dose-response relationship shows a dramatic rightward shift with a K0.5 ⬎50-fold higher than that of wildtype channels. A more conservative mutation (Y1219W),
however, did not change the K0.5 value significantly. The
ATP dose-response relationships of Y1219F and Y1219I
mutants lie between those of wild type and Y1219G, suggesting a correlation between changes of the ATP sensitivity and the chemical natures of the side chain at this
position. Single-channel kinetic analysis indicates that the
shifts of the ATP dose-response relationships in Y1219G
and Y1219I mutants are mainly due to changes of the
opening rate (360). These results are consistent with the
idea that Y1219 mutations change the ATP binding affinity
at the APB2 and confirm the idea that ATP binding at the
ABP2 plays a critical role in catalyzing channel opening
(112, 237, 324). Unlike the mutation at the Walker A lysine
residue, the Y1219G mutation does not affect the opentime constant significantly, suggesting that the mutation
does not alter ATP hydrolysis at ABP2. This latest study
also provides the first piece of evidence that ATP binding
at ABP1 alone (ABP2 is empty) does not increase the
opening rate. This is perhaps not surprising since the
conserved pair of amino acids (R555 and T1246) impor-
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important functional implication of this latest biochemical result is that a complete transport cycle involves not
only ATP hydrolysis at the catalysis-competent site but
also ATP dissociation from the catalysis-incompetent site.
If we consider the ABP2 as a catalytic site for CFTR or
other members of the ABCC family, ABP1 may serve as an
allosteric site that modulates the whole reaction cycle.
Altogether, these results suggest a gating model that
incorporates CFTR’s two ABPs. In the absence of ATP,
the opening rate of CFTR channels is extremely small (33,
35). ATP binds to the low-affinity ABP2 to increase the
rate of channel opening through NBD dimerization (97,
323). For wild-type CFTR, it is the hydrolysis of the bound
ATP at ABP2 that closes the channel. When ATP hydrolysis is abolished by mutations, the channel is locked in a
stable open state; without the free energy from ATP hydrolysis, channel closing through thermal agitation takes
a long time (Fig. 6B). Although it remains controversial
whether ATP binding to ABP1 is required for channel
opening by ATP, data in Zhou et al. (360) suggest that ATP
binding to ABP1 alone fails to increase the opening rate.
Thus the functional asymmetry of the two ATP binding
sites echoes abundant evidence for a biochemical/structural asymmetry of CFTR’s two NBDs described above.
This molecular picture of how ATP binding to two
functionally distinct ABPs induces NBD dimerization predicts different functional anomalies caused by diseaseassociated mutations at each ABP. More than 1,000 different mutations have been identified to cause CF. Relevant to the current article is the gating dysfunction caused
by mutations in ABPs. Most notable are G551D and
G1349D, two mutations located in the signature sequence
of NBD1 (ABP2) and NBD2 (ABP1), respectively. G551D
is the third overall most common CF mutation, with a
worldwide frequency of ⬃3% (www.genet.sickkids.on.ca/
cftr). This mutation is associated with a severe phenotype
characterized by pulmonary dysfunction and pancreatic
insufficiency (63). The G551D mutation results in a significantly decreased chloride current due to a drastic reduction of the channel activity (41, 73, 334, 337). On the
contrary, the G1349D mutation causes a mild form of CF
(271). Although some functional studies have shown that
these two mutant channels exhibit low Po with altered
pharmacological properties (e.g., Ref. 41), it is unclear
how these two mutants respond differently to ATP to
account for the phenotypical difference in patients carrying the mutated genes. Recent studies by Bompadre et al.
(35) indicate that the G551D mutation completely abolishes ATP-dependent gating of CFTR (also see Ref. 329),
whereas G1349D-CFTR retains some ATP dependence
albeit with a Po ⬃10-fold lower than that of WT-CFTR.
These distinct gating defects manifested in mutations at
CFTR’s two signature sequences again resonate the functional asymmetry of the two ABPs in controlling ATPdependent gating of CFTR.
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and AMP-PNP, ABP1 and ABP2 are occupied by ATP and
AMP-PNP, respectively, this finding indicates that P-ATP
stabilizes the open state by binding to ABP1.
The idea that ATP binding at the ABP1 stabilizes the
open state probably also explains the decrease of the
open time by ADP, first discovered by Weinreich et al.
(333) from macroscopic relaxation analysis, and later
confirmed at a single-channel level (33). Also consistent
with this idea, Csanady et al. (59) reported that the removal of part of the “regulatory insertion” in NBD1 does
not affect apparent Kd for ATP, but significantly shortens
the open time. Perhaps the function of this regulatory
insertion is to provide a chemical mechanism for a tighter
binding of ATP at ABP1. It should be noted that the
“regulatory insertion” contains two additional aromatic
amino acids (F409 and F430 in human NBD1) that interact
with the adenine ring of ATP in the crystal structure of
mouse NBD1 (167). Since multiple aromatic amino acids
have been shown to be important in forming a highaffinity binding pocket for nucleotides (103, 133, 134) and
biochemical studies demonstrated that NBD1 assumes a
higher affinity for ATP than NBD2 (10), Zhou et al. (360)
examined the effect of mutations and a combination of
mutations at W401, F409, and F430 residues on the open
state (or NBD dimer) stability under the E1371S background. The locked open time of E1371S-CFTR was shortened in a graded manner as the number of altered aromatic amino acids was increased. Interestingly, this effect
of mutation on the open state can be partly compensated
by using P-ATP, supporting an energetic mechanism instead of nonspecific effects on channel structures. By
applying mutant cycle analyses, Zhou et al. (360) were
able to show that P-ATP binds to ABP1 to prolong the
open time of E1371S-CFTR. It should be noted that this
latest proposition suggests a ready association/dissociation of ATP from ABP1 at room temperature where the
experiments were carried out. This contradicts biochemical results suggesting that ATP is occluded for minutes in
ABP1 (24). More studies are needed to elucidate the
functional significance of this ATP-occluded state.
Irrespective of the duration ATP stays bound in
ABP1, the consequential stabilization of the open state
could provide a distinct advantage for CFTR to function as
an ion channel rather than a transporter since only the open
state conducts ions. On the flip side of the coin, the substrate
transport rate for a transporter is decided by the turnover
rate of a full hydrolysis cycle. If the aforementioned energetic mechanism is applicable to a transporter, decreasing the ATP binding affinity at the catalysis-incompetent
site (i.e., the NH2-terminal NBD) is expected to increase
the substrate transport rate for the transporter in the
ABCC subfamily. A recent report by Yang et al. (344)
demonstrated that the solute transporter rate of MRP1, a
member of the ABCC subfamily, is indeed increased in
mutants whose ATP affinity at NBD1 is decreased. One
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TSUNG-YU CHEN AND TZYH-CHANG HWANG
G. Outstanding Issues and Future Directions
Physiol Rev • VOL
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In the last few years we have witnessed rapid
progress in our structural and functional understanding of
CFTR and other ABC proteins. A dynamic picture of
CFTR channel gating has begun to emerge. This picture
addresses some critical issues in the CFTR field and lays a
new foundation for future studies. At the same time, it also
raises new questions. One obvious question is how ATP
binding catalyzes dimer formation. For instance, after ATP
binds to the Walker A and Walker B of one NBD, how is
the signature sequence of the partner NBD recruited?
While most of the studies of CFTR gating discussed above
focus on residues in the Walker A and Walker B motifs,
very few have tackled the role of signature sequences,
which constitute the complementary partner to form each
ABP. The physiological importance of CFTR’s signature
sequences is demonstrated by the fact that many diseaseassociated mutations are found in these regions. As discussed in section VIIIF, mutations at the conserved glycine
residues in the two signature sequences result in distinct
gating dysfunction. More thorough studies of these mutants are needed to elucidate the functional role of the
signature sequences. This line of research is perhaps even
more critical given that mutating the Walker A and B
motifs has resulted in much conflicting data. It is interesting to note that, although the activity of G551D-CFTR
is ATP independent, 2⬘-deoxy-ATP increases the Po of this
mutant mainly by prolonging the open time (41). How and
where this ATP analog works to enhance the channel
activity are yet to be elucidated. Nevertheless, understanding the fundamental mechanisms responsible for the
defective gating function could pave the way for development of tailored interventions in CF treatment.
Another important area of research is how the R
domain interacts with NBDs to affect the dimer formation. As described above, removal of the R domain renders the channel activity phosphorylation independent,
implying an inhibitory function of an unphosphorylated R
domain. However, the open-time constant of the ⌬RCFTR is slightly smaller than that of wild-type channels
(60). Thus the interaction between NBDs and the R domain may somehow stabilize NBD dimer formation. Recent biochemical studies also demonstrated that phosphorylation promotes not only NBD dimerization (207),
but also the association of the R domain with other domains of CFTR (44). In addition to the well-established
activation role for PKA-dependent phosphorylation, the
demonstration that some of the phosphorylation sites
may inhibit CFTR activity (43, 62, 338) further complicates this already complex issue. Although early studies
suggest that the more than 10 PKA consensus serines in
the R domain may be functionally redundant in activating
the channel (260, 284), it seems important, but quite challenging, to ask how individual phosphorylation sites mod-
ulate these interactions. A new approach to address Rdomain function emerges recently. By comparing NMR
spectra of the R domain in the presence and absence of
NBD1, Baker et al. (22) provided evidence for interactions
between NBD1 and multiple sites in the R domain with
measured fractional helical propensity. This approach together with careful functional studies should propel us
toward a molecular understanding of how the R domain
modulates CFTR function.
A major challenge in understanding CFTR gating is to
relate biochemical events in CFTR’s two ABPs to gating
transitions. Our discussion has been focused on relating
ATP binding/hydrolysis to gating since the gating cycle
(⬃1 s⫺1) is compatible with the measured ATP hydrolysis
rate for purified CFTR (170). However, biochemical studies with isolated NBD2 showed adenylate kinase activity
(107, 253). Using a variety of pharmacological maneuvers,
Randak and Welsh (254) demonstrated a correlation between adenylate kinase activity and CFTR gating function. Furthermore, contrary to previous studies showing
that ADP inhibits CFTR by a competitive inhibition mechanism (17, 33, 281, 333), recent data suggest that ADP
modulates CFTR function by perturbing adenylate kinase
activity (255). Since most of these conclusions were made
based on macroscopic measurements, microscopic kinetic studies that may directly relate adenylate kinase
reactions to gating transitions are needed. Moreover, a
direct demonstration of adenylate kinase activity with the
whole CFTR protein seems critical at this juncture. It
might be worth noting that in most of the studies mentioned here, CFTR gating was observed in excised
patches with no ADP or AMP present. Under such conditions, adenylate kinase activity is hardly conceivable, suggesting that adenylate kinase activity is at least not required for channel gating.
The difficulty of relating biochemical data to functional results is amplified by the fact that these two lines
of studies very often are carried out in different experimental conditions. For example, solutions with a low
ionic strength, commonly used in biochemical studies for
cell lysis (10), may affect gating dramatically (341). Since
CFTR gating is very sensitive to temperature changes (8,
61, 204), low-temperature incubation, a common practice
in biochemical experiments (9, 24, 305), could affect data
interpretations. In addition, the phosphorylation status of
CFTR, a known modulating factor for CFTR gating (96) and
NBD dimerization (207), may not be the same in biochemical experiments as in functional studies. Nevertheless, the
field will benefit tremendously from development of a
readily accessible method for large-scale production of
CFTR proteins suitable for both biochemical and functional investigations.
In spite of a sweeping pace toward a better understanding of the action of NBDs, CFTR’s gating machinery,
numerous puzzling questions remain unanswered. For ex-
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CLC-0 AND CFTR
IX. EVOLUTION AND SPECIATION
By operational definition, both CLC-0 and CFTR, two
structurally unrelated proteins, are ligand-gated chloride
channels. Several biophysical properties, however, distinguish these two proteins from classical ligand-gated ion
channels such as acetylcholine- or GABA-gated ion chanPhysiol Rev • VOL
nels. First, both CLC-0 and CFTR are considered “slow”
channels. The maximal opening rate for CFTR (i.e., at a
saturating [ATP]) is ⬃3 s⫺1, in contrast to ⬃2,000 s⫺1 for
liganded GABA receptor channels (106) and ⬃10,000 s⫺1
for acetylcholine receptor channels (182). Even with the
so-called fast-gating of CLC-0, the opening rate is on the
order of ⬃100 s⫺1, whereas the time constant for the slowgating is in tens to hundreds of seconds (50, 243). Second,
even in the absence of ligand, CFTR channels have a finite
Po of ⬃0.002 (33, 35); ATP binding increases the Po to
0.4 – 0.5, an amplification of ⬃200. Similarly, there exists a
significant basal activity for CLC-0. Ligand increases the
Po by ⬃100-fold (53). On the other hand, the gating equilibrium constant for a doubly-liganded acetylcholine receptor channel can be 107-fold of that for an unliganded
channel (294). Third, while traditional ligand-gated ion
channels follow an allosteric mechanism with kinetic
steps that are in thermodynamic equilibrium, this microscopic reversibility is violated in the gating process of
CLC-0 and CFTR channels because of an input of free
energy. For CLC-0, the gating cycle is coupled to the flow
of Cl⫺ (and/or H⫹). In the case of CFTR, the gating cycle
is driven by ATP hydrolysis. This last biophysical property
not only accounts for a strict stoichiometry of one (or
two) ligand/one gating cycle, but also adequately explains
why these two channels evolved not from the ion channel
families, but from transporter proteins. Capitalizing on
recent breakthroughs in high-resolution structure determination of transporter proteins as well as from sequence
data mining in genomic research and functional insights
from decades of single-channel studies, we intend to speculate daringly here on the potential evolutionary trajectory of transporter-to-channel metamorphosis.
Based on the idea that gene duplication is the major
force driving evolution (228), we reason that the first
primordial ABC transporter is composed of two identical
NBDs and MSDs, encoded by two separate genes. Assembling two MSDs en face in the membrane and two NBDs
in the juxtamembrane cytosol can generate a functional
transporter with a three-dimensional structure similar to
those in Figure 4. Although this ABC protein ancestor
possesses two identical half-molecules (each is equipped
with a transport engine), hydrolysis of one ATP is probably sufficient to complete the transport cycle, since eliminating one ATP hydrolysis site of HisP, an ABC protein
with this primitive structural design, can still sustain the
transport function (226).
If one hydrolysis cycle can effectively fuel the transport cycle, it is not surprising that transporters in the now
ABCC subfamily emerge through “selection-directed mutagenesis” by the invisible force of nature. Members in
this subfamily, including CFTR, contain only one NBD
with the canonical sequences for ATP hydrolysis. Interestingly, many members of the ABCC subfamily serve as
transporters that export organic anions (69). To perform
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ample, how is CFTR function modulated by scores of
proteins shown to interact with CFTR (110, 169)? How
does extracellular [Cl⫺] affect CFTR gating (340)? Is gating control in native tissues different from what has been
unveiled in heterologous expression systems (256, 257)?
It also has been known for years that CFTR gating in
intact cells is very different from that in cell-free systems
such as inside-out membrane patches (119, 139, 350).
Although a clear transition of CFTR gating patterns can be
discerned from cell-attached to excised patches, the fundamental mechanism for this difference in gating is still
unknown. Answering these questions may pose significant impacts on development of therapeutical interventions for CF patients carrying mutations that affect CFTR
gating.
For conformational changes associated with CFTR
gating, immediate questions include the following: Where
is the channel gate? How is the signal transduced from the
NBDs to the channel gate? Is this process similar to those
for classical ligand-gated ion channels such as acetylcholine-gated cation channels (reviewed in Ref. 294), where
many subtle conformational changes spreading from the
ligand binding site to the gate? Recent studies of CFTR
gating energetics indeed suggest a spreading conformational change initiated by NBD dimerization (61). By carefully examining temperature dependence of CFTR gating
through hydrolytic and nonhydrolytic paths, Csanady
et al. (61) discovered large enthalpy and entropy changes
upon channel opening to the transitional state. A large
increase in entropy is consistent with NBD dimerization
that excludes water molecules from the dimer interface,
whereas an even larger enthalpy change indicates molecular strain that is subsequently relieved by opening of the
gate. The crystal structures of ABC transporters discussed in section VII have pointed to several possible
structural motifs that lie between NBDs and MSDs. Future studies of the functional role of these areas could
provide further insight into how NBD dimerization is
coupled to the gate of CFTR. In the next section, we will
try to imagine the evolutionary path from an ABC transporter to the CFTR channel. Stealing the idea from a
recent article by Chris Miller (211), we intend to view
CFTR chloride channel gating through a transporter lens
with the hope that structure/function studies at both ends
will eventually help us understand how transport proteins
work in general.
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TSUNG-YU CHEN AND TZYH-CHANG HWANG
Physiol Rev • VOL
thought to have two “gates.” The side chain of Gluex is
thought to be the external gate. Where is the internal
gate? The constriction of the Cl⫺ transport pathway by
the side chain of S107 may serve as the internal one, but
MTS modification data of Y445C suggest that this constriction does not completely obstruct the transport pathway. The concept of “gate” for ion transport is not necessarily a physical one. A “virtual” gate with an insurmountable energy barrier for ion movement may also
serve the purpose. The observation that Cl⫺ occupancy at
Scen is critical for H⫹ transport is suggestive. If Cl⫺ occupancy at Scen is absolutely required for the H⫹ transport,
the Cl⫺-Scen complex can serve as a dynamic internal
gate. When Cl⫺ occupies at Scen, the gate is open for H⫹
transport. Without Cl⫺ occupancy at this site, H⫹ cannot
travel to this site as if the gate is closed for H⫹. When Cl⫺,
H⫹, and Scen form a complex, the external gate is closed.
Once H⫹ travels to Gluex to open this external gate, the
internal virtual gate is closed because Cl⫺ may leave Scen
if Cl⫺ and H⫹ bindings are synergistic. Thus there does
not exist a moment when both gates are open, an essential feature for the action of transporters.
The speculated change during evolution from primordial ABC transporter molecules to CFTR Cl⫺ channels
involves significant structural alterations of the protein
molecule to remove one of the two gates. However, for
CLC family members, the evolution from CLC antiporter
to CLC Cl⫺ channels may involve more subtle changes if
the speculated CLC antiport mechanism described above
is correct. To remove one gate, uncoupling the Cl⫺ and
H⫹ transport can serve the purpose. Blocking the H⫹
transport, as seen in the mutational studies of Gluex and
Gluin, has converted the bacterial antiporter into a Cl⫺
uniporter (or Cl⫺ channel). For CLC members, the residue that corresponds to Glu-203 of CLC-ec1 (the Gluin) is
no longer a protonatable residue in channel members, but
a glutamate residue is conserved in the members that are
thought to be transporters. It seems difficult to imagine
that evolution converts a transporter into an ion channel
by a single amino acid mutation, but such a relation
between sequence analysis and functional classification is
striking. Perhaps more extensive structural alterations
are involved, and more theoretical and experimental work
is needed to deepen our understanding of the structural/
functional relationship between ion channels and transporters in this family. The residue corresponding to Glu203 of CLC-ec1 and its surrounding region may serve as a
launch platform for exploration. Clearly, most of the speculations made above will not stand the scrutiny of future
work. We believe, however, that defining what makes a
channel a channel and a transporter a transporter will
continue to be an interesting puzzle. As more crystal
structures of both CLC and ABC proteins are solved and
more functional data are gathered, a clearer picture of
transporter-channel ties is expected to come to light.
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this function, the ABC proteins in this subfamily should
be equipped with high-affinity anion binding site(s) facing
the cytoplasmic side of the transport pathway. In addition, the “rocking banana” model (Fig. 1C) dictates that
the anion-binding site needs to be sandwiched by two
physical gates. Suppose one of these two gates is degraded in the process of speciation; NBD dimerization is
now coupled to movement of only one gate that guards
the negatively charged substrate bound in the transport
pathway (which now can be called a “pore”). As described
above, using organic anionic blockers as probes, numerous studies suggest the presence of an internal vestibule
in the CFTR pore (reviewed by Linsdell, Ref. 178). Some
results also point to a physical gate, located at the cytoplasmic end of the CFTR pore that controls the accessibility of the blocker binding site (e.g., Ref. 289). Among
the numerous blockers, glibenclamide provides unique
insights because both the on- and off-rates of this blocker
can be measured at the single-channel level (356). The
extraordinarily high voltage-dependent binding and unbinding of the gigantic glibenclamide (21.4 Å ⫻ 13.6 Å ⫻
11.6 Å) in the CFTR pore suggest the presence of a wide
and deep vestibule accessible from the cytoplasmic side
of the channel (289, 356). From a teleological point of
view, it seems difficult to rationalize the raison d’être for
this vestibule where many intracellular organic anions
can potentially plug the pore and prevent Cl⫺ diffusion.
Perhaps this functionally defined vestibule represents an
evolutionary remnant.
Admittedly, this naive conjecture has a few holes.
First, there is only limited evidence that CFTR can
conduct bulky organic anions (180). In fact, those organic blockers are so named because they are impermeant. However, this stain can be removed by imagining a selectivity filter built at the distal end of the
blocker binding site as proposed by Zhou et al. (356).
Second, based on the model proposed by Locher and
colleagues (128), ATP binding-induced NBD dimerization actually closes the cytoplasmic access to the transport pathway. This is in direct conflict with the fact that
ATP-induced dimerization of CFTR’s NBDs opens the
gate of the channel. Third, if the degraded-gate idea is
correct, CFTR can no longer serve as a transporter.
However, recent studies by Kogan et al. (158) suggest a
possible transporter function of CFTR. There is still
much to learn about the fascinating and puzzling relationship between ion channels and transporters. Solving the high-resolution crystal structure of the whole
CFTR protein, though a mounting challenge at this
juncture, will definitely illuminate our quest.
In the CLC family, structural data have argued that
the structure of the transporter CLC-ec1 does not undergo
a large conformational change when the molecule carries
out its function, a situation very different from the general
antiporter model. As described above, a transporter is
379
CLC-0 AND CFTR
ACKNOWLEDGMENTS
We are indebted to our colleagues for their constructive
criticisms and inspirational discussions. We are grateful to Drs.
Joseph Mindell and David Sheppard for their critical reading of
the manuscript. We also thank Drs. Chris Miller and Jie Zheng
for providing pictures that are converted to some of the figures.
Address for reprint requests and other correspondence:
T. -Y. Chen, Center for Neuroscience, Univ. of California, Davis,
1544 Newton Ct., Davis, CA 95618 (e-mail: tycchen@ucdavis.
edu).
17.
18.
19.
20.
21.
GRANTS
22.
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Ionic currents mediated by a prokaryotic homologue of CLC Cl⫺
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