The opening and closing of chloride (Cl-) channels in the ClC family are thought to tightly couple to ion permeation through the channel pore. In the prototype channel of the family, the ClC-0 channel from the Torpedo electric organ, the opening-closing of the pore in the millisecond time range known as "fast gating" is regulated by both external and internal Cl- ions. Although the external Cl- effect on the fast-gate opening has been extensively studied at a quantitative level, the internal Cl- regulation remains to be characterized. In this study, we examine the internal Cl- effects and the electrostatic controls of the fast-gating mechanism. While having little effect on the opening rate, raising [Cl-]i reduces the closing rate (or increases the open time) of the fast gate, with an apparent affinity of >1 M, a value very different from the one observed in the external Cl- regulation on the opening rate. Mutating charged residues in the pore also changes the fast-gating properties-the effects are more prominent on the closing rate than on the opening rate, a phenomenon similar to the effect of [Cl-]i on the fast gating. Thus, the alteration of fast-gate closing by charge mutations may come from a combination of two effects: a direct electrostatic interaction between the manipulated charge and the negatively charged glutamate gate and a repulsive force on the gate mediated by the permeant ion. Likewise, the regulations of internal Cl- on the fast gating may also be due to the competition of Cl- with the glutamate gate as well as the overall more negative potential brought to the pore by the binding of Cl-. In contrast, the opening rate of the fast gate is only minimally affected by manipulations of [Cl-]i and charges in the inner pore region. The very different nature of external and internal Cl- regulations on the fast gating thus may suggest that the opening and the closing of the fast gate are not microscopically reversible processes, but form a nonequilibrium cycle in the ClC-0 fast-gating mechanism.

ClC-0 is a chloride channel whose gating is sensitive to both voltage and chloride. Based on analysis of gating kinetics using single-channel recordings, a five-state model was proposed to describe the dependence of ClC-0 fast-gate opening on voltage and external chloride (Chen, T.-Y., and C. Miller. 1996. J. Gen. Physiol. 108:237-250). We aimed to use this five-state model as a starting point for understanding the structural changes that occur during gating. Using macroscopic patch recordings, we were able to reproduce the effects of voltage and chloride that were reported by Chen and Miller and to fit our opening rate constant data to the five-state model. Upon further analysis of both our data and those of Chen and Miller, we learned that in contrast to their conclusions, (a) the features in the data are not adequate to rule out a simpler four-state model, and (b) the chloride-binding step is voltage dependent. In order to be able to evaluate the effects of mutants on gating (described in the companion paper, see Engh et al. on p. 351 of this issue), we developed a method for determining the error on gating model parameters, and evaluated the sources of this error. To begin to mesh the kinetic model(s) with the known CLC structures, a model of ClC-0 was generated computationally based on the X-ray crystal structure of the prokaryotic homolog ClC-ec1. Analysis of pore electrostatics in this homology model suggests that at least two of the conclusions derived from the gating kinetics analysis are consistent with the known CLC structures: (1) chloride binding is necessary for channel opening, and (2) chloride binding to any of the three known chloride-binding sites must be voltage dependent.

Clinical and epidemiological data have associated chronic inflammation with cancer progression. Most tumors show evidence of infiltrating immune and inflammatory cells, and chronic inflammatory disorders are known to increase the overall risk of cancer development. While immune cells are often observed in early hyperplastic lesions in vivo, there remains debate over whether these immune cells and the cytokines they produce in the developing hyperplastic microenvironment act to inhibit or facilitate tumor development. The interleukin-6 (IL-6) family of cytokines, which includes IL-6 and oncostatin M (OSM), among others (LIF, CT-1, CNTF, and CLC), are secreted by immune cells, stromal cells, and epithelial cells, and regulate diverse biological processes. Each of the IL-6 family cytokines signals through a distinct receptor complex, yet each receptor complex uses a shared gp130 subunit, which is critical for signal transduction following cytokine binding. Activation of gp130 results in the activation of Signal Transducer and Activator of Transcription 3 (STAT3), and the Mitogen-Activated Protein Kinase (MAPK) and Phosphatidylinositol 3-Kinase (PI3K) signaling cascades. Tumor suppressive signaling can often be observed in normal cells following prolonged STAT3 activation. However, there is mounting evidence that the IL-6 family cytokines can contribute to later stages of tumor progression in many ways. Here we will review how the microenvironmental IL-6 family cytokine OSM influences each stage of the transformation process. We discuss the intrinsic adaptations a developing cancer cell must make in order to tolerate and circumvent OSM-mediated growth suppression, as well as the OSM effectors that are hijacked during tumor expansion and metastasis. We propose that combining current therapies with new ones that suppress the signals generated from the tumor microenvironment will significantly impact an oncologist's ability to treat cancer.

Although multihost complex life cycles (CLCs) are common in several distantly related groups of parasites, their evolution remains poorly understood. In this article, we argue that under particular circumstances, adding a second host to a single-host life cycle is likely to enhance transmission (i.e., reaching the target host). For instance, in several situations, the propagules of a parasite exploiting a predator species will achieve a higher host-finding success by encysting in a prey of the target predator than by other dispersal modes. In such a case, selection should favor the transition from a single- to a two-host life cycle that includes the prey species as an intermediate host. We use an optimality model to explore this idea, and we discuss it in relation to dispersal strategies known among free-living species, especially animal dispersal. The model found that selection favored a complex life cycle only if intermediate hosts were more abundant than definitive hosts. The selective value of a complex life cycle increased with predation rates by definitive hosts on intermediate hosts. In exploring trade-offs between transmission strategies, we found that more costly trade-offs made it more difficult to evolve a CLC while less costly trade-offs between traits could favor a mixed strategy.

CLC-type chloride channels exhibit a unique double-barreled architecture with two independently functioning ion conduction pathways, the so-called protopores. There exist gating processes that open and close individual protopores as well as common processes that jointly mediate slow opening and closing of both protopores. Different isoforms exhibit distinct voltage dependences and kinetics of gating. Whereas opening of the individual and common gate of homo-dimeric ClC-1 is promoted by membrane depolarization, ClC-2 is closed at positive potentials and opens only at negative voltages. To characterize the functional interaction of protopores we engineered a concatameric construct linking the coding regions of ClC-1 and ClC-2 in an open reading frame, expressed it in mammalian cells and measured anion currents through whole-cell and single channel patch clamping. In the hetero-dimeric assembly, each protopore displayed two kinetically distinct gating processes. Fast gating of the ClC-1 protopore closely resembled fast protopore gating of homo-dimeric channels. The voltage dependence of ClC-2 fast gating was shifted to more positive potentials by the adjacent ClC-1 protopore, resulting in open ClC-2 protopores at positive voltages. We observed two slow gating processes individually acting on ClC-1 and ClC-2 protopores, with distinct time and voltage dependences. Single channel recordings demonstrated that hetero-dimerization additionally modified the unitary conductance of ClC-2 protopores. Our findings suggest that inter-subunit interactions do not only affect common gating, but also ion permeation and gating of individual protopores in hetero-dimeric ClC channels.

Chloride channels are widely expressed and play important roles in cell volume regulation, transepithelial transport, intracellular pH regulation, and membrane excitability. Most chloride channels have yet to be identified at a molecular level. The ClC gene family and the cystic fibrosis transmembrane conductance regulator (CFTR) are distinct chloride channels expressed in many cell types, and mutations in their genes are the cause of several diseases including myotonias, cystic fibrosis, and kidney stones. Because of their molecular definition and roles in disease, these channels have been studied intensively over the past several years. The focus of this review is on recent studies that have provided new insights into the mechanisms governing the opening and closing, i.e. gating, of the ClC and CFTR chloride channels.

BACKGROUND

Dent's disease, a renal tubular disorder characterized by low-molecular-weight proteinuria, hypercalciuria, and nephrolithiasis, is due to inactivating mutations in the X-linked renal-specific chloride channel, hCLC-5. The x-ray crystal structures of two bacterial chloride channels (CLCs) have recently been established, thereby allowing us to construct a model for hCLC-5 and further examine the role of its mutations.

METHODS

The data regarding 49 hCLC-5 mutations were reviewed. Thirty-four mutations that predicted absent or truncated channels were excluded. The remaining 15 mutations (one in-frame insertion and 14 missense mutations), 12 of which have been studied electrophysiologically, were assessed. The hCLC-5 sequence was aligned with the Salmonella typhimurium and Escherichia coli sequences and used to map the hCLC-5 mutations onto a three-dimensional model.

RESULTS

hCLC-5 is a homodimeric protein, with each subunit consisting of 18 helices. None of the missense mutations involved the chloride (Cl-) selectivity filter, but 12 of the 15 mutations were found to be clustered at the interface of the two subunits. Six of these mutations occurred in two of the helices that either form part of the interface or lie in close proximity to the interface, and three other mutations that did not lead to complete loss of Cl- conductance were at the edge of the interface.

CONCLUSIONS

These results demonstrate a crucial role for the interaction between the two subunits at the interface of the homodimeric hCLC-5.

Auditory feedback is critical for the development and maintenance of speech in humans. In contrast, studies of nonhuman primate vocal production generally report that subjects show little reliance on auditory input. We examined the extent to which cotton-top tamarin (Saguinus oedipus) vocal production is sensitive to perturbation of auditory feedback by manipulating the predictability of presentation of a 1 s burst of white noise during the production of the species-specific contact call, the combination long call (CLC). We used three experimental conditions: the Begin condition, in which white noise was presented only during the first half of a recording session, the End condition, in which white noise was presented only in the last half, and the Random condition, in which each call had a 50% probability of receiving white noise playback throughout the recording session, making the auditory feedback unpredictable. In addition we recorded calls before and after the experimental series (Baseline condition) to determine whether any changes induced by modification of auditory feedback persisted. Results showed that playback of white noise during the production of the CLC produced changes in the temporal structure of the CLC: calls were shorter and had fewer pulses, indicating that modification of auditory feedback can interrupt vocal production. In addition, calls that received modified feedback were louder and had longer inter-pulse intervals than those that did not, consistent with an adaptive response to the masking effect of white noise playback. The magnitude of this compensatory effect and the interruption rate were both sensitive to whether the feedback modification occurred at the beginning or end of the experimental session: early feedback produced less interruption and more compensation. Finally, when auditory feedback modification was unpredictable, adaptive changes were observed in both calls that received modified feedback and those that received normal feedback, suggesting that tamarins can generate an expectation of noise playback and increase vocal amplitude in anticipation of masking.

Pancreatic duct cells express a Ca2+-activated Cl- conductance (CaCC), upregulation of which may be beneficial to patients with cystic fibrosis. Here, we report that HPAF, a human pancreatic ductal adenocarcinoma cell line that expresses CaCC, develops into a high-resistance, anion-secreting epithelium. Mucosal ATP (50 microM) caused a fourfold increase in short-circuit current (Isc), a hyperpolarization of transepithelial potential difference (from -4.9 +/- 0.73 to -8.5 +/- 0.84 mV), and a fall in resistance to less than one-half of resting values. The effects of ATP were inhibited by mucosal niflumic acid (100 microM), implicating an apical CaCC in the response. RT-PCR indicated expression of hClC-2, hClC-3, and hClC-5, but surprisingly not hCLCA-1 or hCLCA-2. K+ channel activity was necessary to maintain the ATP-stimulated Isc. Using a pharmacological approach, we found evidence for two types of K+ channels in the mucosal and serosal membranes of HPAF cells, one activated by chlorzoxazone (500 microM) and sensitive to clotrimazole (30 microM), as well as one blocked by clofilium (100 microM) but not chromanol 293B (5 microM). RT-PCR indicated expression of the Ca2+-activated K+ channel KCNN4, as well as the acid-sensitive, four transmembrane domain, two pore K+ channel, KCNK5 (hTASK-2). Western blot analysis verified the expression of CLC channels, as well as KCNK5. We conclude that HPAF will be a useful model system for studying channels pertinent to anion secretion in human pancreatic duct cells.

Vocal production can be highly deterministic, such that once the central nervous system generates a signal to call, the vocalization is emitted immune to external events. Conversely, vocal production can be modulated by auditory feedback such that interference or disruption can cause an individual to stop calling or, if it continues to call, for the acoustic morphology of the signal to change. To explore which of these models best accounts for the control of vocal production in non-human primates, we adapted an interruption technique originally developed for songbirds for use with a New World monkey species, the cotton-top tamarin (Saguinus oedipus). Results from a pilot experiment indicated that an auditory stimulus (white noise) was more effective than a visual stimulus (strobe light) at interrupting the tamarin's species-typical 'combination long call (CLC)'. Data from a second experiment showed that although the duration of the auditory stimulus did not affect the proportion of interruptions that occurred, a 1000 ms white noise stimulus perturbed the temporal structure of the CLC to a greater extent than did a 250 ms white noise stimulus. Furthermore, when call production was interrupted, tamarins stopped vocalizing after the completion of a syllable, suggesting that the syllable represents a unit of organization within the call. Overall, these results provide evidence that tamarins can modify their vocal output based on external events, but the degree of vocal control is significantly less than in oscine songbirds.

The study of ion channel function is constrained by the availability of structures for only a small number of channels. A commonly used bioinformatics technique is to assert, based on sequence similarity, that a domain within a channel of interest has the same structure as a reference domain for which the structure is known. This technique, while useful, is often employed when there is only a slight similarity between the channel of interest and the domain of known structure. In this study, we exploit recent advances in structural genomics to calculate the sequence-based probability of the presence of putative domains in a number of ion channels. We find strong support for the presence of many domains that have been proposed in the literature. For example, eukaryotic and prokaryotic CLC proteins almost certainly share a common structure. A number of proposed domains, however, are not as well supported. In particular, for the COOH terminus of the BK channel we find a number of literature proposed domains for which the assertion of common structure based on common sequence has a nontrivial probability of error.

Osteoarthritis (OA) is the most prevalent joint disease and is characterized by pain and functional loss of the joint. However, the pathogenic mechanism of OA remains unclear, and no drug therapy for preventing its progress has been established. To identify genes related to the progress of OA, the gene expression profiles of paired intact and damaged cartilage obtained from OA patients undergoing joint substitution were compared using oligo microarrays. Using functional categorization combined with gene ontology and a statistical analysis, five genes were found to be highly expressed in damaged cartilage (HBEGF, ASUS, CRLF1, LOX, CDA), whereas three genes were highly expressed in intact tissues (CHST2, PTPRD, CPAN6). Among these genes, the upregulated expression of CRLF1 was reconfirmed using real-time PCR, and the in vivo expression of CRLF1 was detected in clusters of chondrocytes and fibrocartilage-like cells in damaged OA cartilages using in situ hybridization. In vitro, the transcriptional level of CRLF1 was positively regulated by TGF-beta1 in the mouse chondrogenic cell line ATDC5. Additionally, the CRLF1/CLC complex promoted the proliferation of ATDC5 cells and suppressed the expression level of aggrecan and type II collagen. Our data suggest that the CRLF1/CLC complex disrupts cartilage homeostasis and promotes the progress of OA by enhancing the proliferation of chondrocytes and suppressing the production of cartilage matrix. A component of the complex, CRLF1, may be useful as a biomarker of OA; and the corresponding receptor is a potential new drug target for OA.

Transport of water and electrolytes is critical for corneal clarity. Recent studies indicate another important function of transport of ions and electrolytes - establishing wound electric fields that guide cell migration. We found chloride (Cl(-)) flux is a major component of the corneal wound electric current. In order to elucidate the mechanisms of Cl(-) transport, we studied Cl(-) channels and transporters in human corneal epithelial (HCE) cells. We tested a transformed human corneal epithelial cell line (tHCE), primary cultures of human corneal epithelial cells (pHCE), and human donor corneas. We first used RT-PCR to determine expression levels of mRNA of CLC (Cl(-) channels/transporters of CLC gene family) family members and CFTR (cystic fibrosis transmembrane conductance regulator) in HCE cells. We then confirmed protein expression and distribution of selected CLC family members and CFTR with Western blot and immunofluorescence confocal microscopy. Finally, Cl(-) currents were recorded with electrophysiological techniques. The mRNAs of CLC-2, CLC-3, CLC-4, CLC-5, CLC-6, and CFTR were detected in the HCE cell line. CLC-1 and CLC-7 were not detectable. Western blot and immunostaining confirmed protein expression and distribution of CLC-2, CLC-3, CLC-4, CLC-6 and CFTR in human corneal epithelium. CLC-2 preferentially labeled the apical and basal layers, while CLC-3 and CLC-4 labeled only the superficial layer. CLC-6 and CFTR labeling showed a unique gradient with strong staining in apical layers which gradually decreased towards the basal layers. Corneal endothelium was positive for CLC-2, CLC-3, CLC-4, CLC-6 and possibly CFTR. Human corneal epithelial cells demonstrated voltage dependent Cl(-) currents. HCE cells express functional Cl(-) channels and transporters. CLC-2, CLC-3, CLC-4, CLC-6, and CFTR had distinct expression patterns in human corneal epithelium. Those molecules and their distribution may play important roles in maintaining resting Cl(-) fluxes and in regulating Cl(-) flux at corneal wounds, which may be a major contributor to wound electrical signaling.

Glycated hemoglobin is widely used in the management of diabetes mellitus. At least 300,000 Americans with diabetes mellitus have the hemoglobin (Hb) C or S trait. The accuracy of HbA1c methods can be adversely affected by the presence of these traits. We evaluated the effects of HbC and HbS traits on the results of 14 commercial HbA1c methods that use boronate affinity, enzymatic, immunoassay, and ion exchange methods. Whole blood samples from people homozygous for HbA or heterozygous for HbC or HbS were analyzed for HbA1c. Results for each sample type were compared with those from the CLC 330 comparative method (Primus Diagnostics, Kansas City, MO). After correcting for calibration bias by comparing results from the homozygous HbA group, method bias attributable to the presence of HbC or HbS trait was evaluated with a clinically significant difference being more than 10% (ie, 0.6% at 6% HbA1c). One immunoassay method exhibited clinically significant differences owing to the presence of HbC and HbS traits.

The genome of the nematode Caenorhabditis elegans encodes six putative chloride channels (CeCLC-1 through CeCLC-6) that represent all three known branches of the mammalian CLC gene family. Using promoter fragments to drive the expression of the green fluorescent protein, CeCLC-2, -3, and -4 expression was studied in transgenic C. elegans. CeCLC-4 was specifically expressed in the large H-shaped excretory cell, where it was co-expressed with CeCLC-3, which is also expressed in other cells, including neurons, muscles, and epithelial cells. Also, CeCLC-2 was expressed in several cells of the nervous system, intestinal cells, and vulval muscle cells. Similar to mammalian CLC proteins, only two nematode CLC channels elicited detectable plasma membrane currents in Xenopus oocytes. CeCLC-3 currents were inwardly rectifying and were activated by positive prepulses. Its complex gating behavior can be explained by two gates, at least one of which depends on extracellular anions. In this respect it resembles some mammalian chloride channels with which it also shares a preference of chloride over iodide. C. elegans thus provides new opportunities to understand common mechanisms underlying structure and function in CLC channels and will allow for a genetic dissection of chloride channels in this simple model organism.

Fetal alcohol syndrome includes brain damage from aberrant synaptogenesis, altered cell-cell signaling and blunted plasticity in surviving neurons. Distortion of neurotrophic GABA signals by ethanol-mediated allosteric modulation of GABA(A) receptor (GABA(A)R) activity during brain maturation may play a role. In this regard, early postnatal binge-like ethanol treatment on postnatal days (PDs) 4-9 acutely inhibits whole cell GABA(A)R Cl(-) current and subsequently blunts GABA(A)R function in medial septum/diagonal band (MS/DB) neurons and cerebellar Purkinje cells [Dev. Brain Res. 130 (2001) 25-40; Brain Res. 810 (1998) 100-113; Brain Res. 832 (1999) 124-135]. In light of these functional changes, we hypothesized that ethanol treatment also would decrease levels of proteins important for assembly of GABAergic synapses in maturing brain. To test this relationship, binge-like ethanol intubation was administered to rat pups on PDs 4-9 producing peak blood ethanol concentrations in the range of 302.5+/-6.3 mg/dl. GABAergic synaptic proteins were measured in brain tissue on PDs 13-14 when GABA(A)R currents in individual MS/DB neurons are reduced, but those of cerebellar Purkinje neurons are not yet altered [Dev. Brain Res. 130 (2001) 25-40; Brain Res. 810 (1998) 100-113; Brain Res. 832 (1999) 124-135]. Surprisingly, ethanol did not decrease protein levels of GABA(A)R alpha1/beta2 subunits, GAD(67) or gephyrin in MS/DB at this time when whole cell recordings indicate GABA(A)R function is impaired in acutely dissociated individual neurons. However, in cerebellum where ethanol treated Purkinje cell GABA(A)R function remains normal on PDs 13-14 [Brain Res. 832 (1999) 124-135], reduced levels of several GABAergic synaptic proteins including: GAD(67), GABA(A)R alpha1 subunit, ClC-2 a voltage-gated Cl(-) channel, synaptotagmin a synaptic vesicle protein, and N-cadherin, a synapse associated cell adhesion molecule, were found. These results indicate that binge-like ethanol exposure differentially decreases GABAergic synaptic proteins in some brain areas in a pattern that does not parallel reductions in GABA(A)R function of individual neurons that survive this ethanol insult.

Early crystal structures of prokaryotic CLC proteins identified three Cl(-) binding sites: internal (S(int)), central (S(cen)), and external (S(ext)). A conserved external GLU (GLU(ex)) residue acts as a gate competing for S(ext). Recently, the first crystal structure of a eukaryotic transporter, CmCLC, revealed that in this transporter GLU(ex) competes instead for S(cen). Here, we use molecular dynamics simulations to investigate Cl(-) transport through CmCLC. The gating and Cl(-)/H(+) transport cycle are inferred through comparative molecular dynamics simulations with protonated and deprotonated GLU(ex) in the presence/absence of external potentials. Adaptive biasing force calculations are employed to estimate the potential of mean force profiles associated with transport of a Cl(-) ion from S(ext) to S(int), depending on the Cl(-) occupancy of other sites. Our simulations demonstrate that protonation of GLU(ex) is essential for Cl(-) transport from S(ext) to S(cen). The S(cen) site may be occupied by two Cl(-) ions simultaneously due to a high energy barrier (∼8 Kcal/mol) for a single Cl(-) ion to translocate from S(cen) to S(int). Binding two Cl(-) ions to S(cen) induces a continuous water wire from S(cen) to the extracellular solution through the side chain of the GLU(ex) gate. This may initiate deprotonation of GLU(ex), which then drives the two Cl(-) ions out of S(cen) toward the intracellular side via two putative Cl(-) transport paths. Finally, a conformational cycle is proposed that would account for the exchange stoichiometry.

Claudins ( approximately 23 kDa) with four transmembrane domains are major cell adhesion molecules working at tight junctions in vertebrates, where the intercellular space is tightly sealed (reviewed in ). We examined here the possible occurrence of claudin-like proteins in invertebrates, which do not bear typical tight junctions. Close blast searching of the C. elegans genome database identified four claudin-related, approximately 20-kDa integral membrane proteins (CLC-1 to -4), which showed sequence similarity to the vertebrate claudins. The expression and distribution of CLC-1 was then examined in detail by GFP technology as well as by immunofluorescence microscopy. CLC-1 was mainly expressed in the epithelial cells in the pharyngeal region of digestive tubes and colocalized with AJM-1 at their intercellular junctions. Then, to examine the possible involvement of CLC-1 in the barrier function, we performed RNA interference in combination with a tracer experiment: in CLC-1-deficient worms, the barrier function of the pharyngeal portion of the digestive tubes appeared to be severely affected. CLC-2 was expressed in seam cells in the hypodermis, and it also appeared to be involved in the hypodermis barrier. These findings indicated that multiple species of the claudin homologs, which are involved in the barrier function of the epithelium, exist in C. elegans.

Anion-transport proteins are central to all of physiology, for processes ranging from regulating bone-density, muscle excitability, and blood pressure, to facilitating extreme-acid survival of pathogenic bacteria. 4,4-Diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) has been used as an anion-transport inhibitor for decades. In this study, we demonstrate that polythiourea products derived from DIDS hydrolysis inhibit three different CLC chloride-transport proteins, ClC-ec1, ClC-0, and ClC-Ka, more effectively than DIDS itself. The structures of the five major products were determined by NMR spectroscopy, mass spectrometry, and chemical synthesis. These compounds bind directly to the CLC proteins, as evidenced by the fact that inhibition of ClC-0 occurs only from the intracellular side and inhibition of ClC-Ka is prevented by the point mutation N68D. These polythioureas are the highest affinity inhibitors known for the CLCs and provide a new class of chemical probes for dissecting the molecular mechanisms of chloride transport.

Members of the CLC 'chloride channel' family play vital roles in a wide variety of physiological settings. Research on prokaryotic CLC homologues provided long-anticipated high-resolution structures as well as the unexpected discovery that some CLCs are not chloride channels, but rather are proton-chloride antiporters. Hence, CLCs encompass two functional classes of transport proteins once thought to be fundamentally different from one another. In this review, we discuss the structural features and molecular mechanisms of CLC channels and antiporters. We focus on ClC-0, the most thoroughly studied CLC channel, and ClC-ec1, the prokaryotic antiporter of known structure. We highlight some striking similarities between these CLCs and discuss compelling questions that remain to be addressed. Prokaryotic CLCs will undoubtedly continue to shed light upon this understudied family of proteins.

The physiologically indispensable chloride channel (CLC) family is split into two classes of membrane proteins: chloride channels and chloride/proton antiporters. In this article we focus on the relationship between these two groups and specifically review the role of protons in chloride-channel gating. Moreover, we discuss the evidence for proton transport through the chloride channels and explore the possible pathways that the protons could take through the chloride channels. We present results of a mutagenesis study, suggesting the feasibility of one of the pathways, which is closely related to the proton pathway proposed previously for the chloride/proton antiporters. We conclude that the two groups of CLC proteins, although in principle very different, employ similar mechanisms and pathways for ion transport.

Secretion of K(+) into endolymph depends on a particular constellation of ion transport proteins in the apical and basolateral membranes of strial marginal cells and vestibular dark cells. One fundamental component is the large chloride conductance of the basolateral membrane, which recycles chloride taken up by the Na(+)-K(+)-Cl(-) cotransporter in the same membrane. Evidence has been reported recently that supports ClC-K, a channel subunit previously thought to be specific to the kidney, as being the molecular entity underlying this conductance. We have isolated protein from the gerbil kidney, stria vascularis and vestibular labyrinth and found by Western blot analysis a 60 kDa band, a 48 kDa band and 54 and 70 kDa bands, respectively, specifically labeled by ClC-K antibody. Subsequent immunohistochemical observations of the inner ear tissues with a confocal microscope on fluorescently labeled tissue sections showed the staining to be restricted to the basolateral region of strial marginal cells and vestibular dark cells. The cochlear staining was distinct from the distribution of the Kir4.1 (KCNJ10) K(+) channel, known to be present only in strial intermediate cells. These findings support the contention that ClC-K is an important component of the basolateral Cl(-) conductance that participates in K(+) secretion by these epithelia.

Dent's disease is an X-linked recessive disorder affecting the proximal tubules and is frequently associated with mutations in CLCN5, which encodes the electrogenic chloride-proton exchanger ClC-5. To better understand the functional consequences of CLCN5 mutations in this disease, we screened four newly identified missense mutations (G179D, S203L, G212A, L469P), one new nonsense mutation (R718X), and three known mutations (L200R, C219R, and C221R), in Xenopus laevis oocytes and HEK293 cells expressing either wild-type or mutant exchanger. A type-I mutant (G212A) trafficked normally to the cell surface and to early endosomes, underwent complex glycosylation at the cell surface like wild-type ClC-5, but exhibited significant reductions in outwardly rectifying ion currents. The type-II mutants (G179D, L200R, S203L, C219R, C221R, L469P, and R718X) were improperly N-glycosylated and were non-functional due to retention in the endoplasmic reticulum. Thus these mutations have distinct mechanisms by which they could impair ClC-5 function in Dent's disease.

CLC-5 is a H(+)/Cl(-) exchanger that is expressed primarily in endosomes but can traffic to the plasma membrane in overexpression systems. Mutations altering the expression or function of CLC-5 lead to Dent's disease. Currents mediated by this transporter show extreme outward rectification and are inhibited by acidic extracellular pH. The mechanistic origins of both phenomena are currently not well understood. It has been proposed that rectification arises from the voltage dependence of a H(+) transport step, and that inhibition of CLC-5 currents by low extracellular pH is a result of a reduction in the driving force for exchange caused by a pH gradient. We show here that the pH dependence of CLC-5 currents arises from H(+) binding to a single site located halfway through the transmembrane electric field and driving the transport cycle in a less permissive direction, rather than a reduction in the driving force. We propose that protons bind to the extracellular gating glutamate E211 in CLC-5. It has been shown that CLC-5 becomes severely uncoupled when SCN(-) is the main charge carrier: H(+) transport is drastically reduced while the rate of anion movement is increased. We found that in these conditions, rectification and pH dependence are unaltered. This implies that H(+) translocation is not the main cause of rectification. We propose a simple transport cycle model that qualitatively accounts for these findings.