Low single channel conductance of the major skeletal muscle chloride channel, ClC-1.

We expressed the skeletal muscle chloride channel, ClC-1, in HEK293 cells and investigated it with the patch-clamp technique. Macroscopic properties are similar to those obtained after expression in Xenopus oocytes, except that faster gating kinetics are observed in mammalian cells. Nonstationary noise analysis revealed that both rat and human ClC-1 have a low single channel conductance of about 1 pS. This finding may explain the lack of single-channel data for chloride channels from skeletal muscle despite its high macroscopic chloride conductance.     

Pores formed by single subunits in mixed dimers of different CLC chloride channels

CLC chloride channels comprise a gene family with nine mammalian members. Probably all CLC channels form homodimers, and some CLC proteins may also associate to heterodimers. ClC-0 and ClC-1, the only CLC channels investigated at the single-channel level, display two conductances of equal size which are thought to result from two separate pores, formed individually by the two monomers. We generated concatemeric channels containing one subunit of ClC-0 together with one subunit of ClC-1 or ClC-2. They should display two different conductances if one monomer were sufficient to form one pore. Indeed, we found a 8-picosiemens (pS) conductance (corresponding to ClC-0) that was associated with either a 1.8-pS (ClC-1) or a 2.8-pS (ClC-2) conductance. These conductances retained their typical gating, but the slow gating of ClC-0 that affects both pores simultaneously was lost. ClC-2 and ClC-0 current components were modified by point mutations in the corresponding subunit. The ClC-2 single pore of the mixed dimer was compared with the pores in the ClC-2 homodimer and found to be unaltered. We conclude that each monomer individually forms a gated pore. CLC dimers in general must be imagined as having two pores, as shown previously for ClC-0.     

Cysteine modification of a putative pore residue in ClC-0: implication for the pore stoichiometry of ClC chloride channels

The ClC channel family consists of chloride channels important for various physiological functions. Two members in this family, ClC-0 and ClC-1, share approximately 50-60% amino acid identity and show similar gating behaviors. Although they both contain two subunits, the number of pores present in the homodimeric channel is controversial. The double-barrel model proposed for ClC-0 was recently challenged by a one-pore model partly based on experiments with ClC-1 exploiting cysteine mutagenesis followed by modification with methanethiosulfonate (MTS) reagents. To investigate the pore stoichiometry of ClC-0 more rigorously, we applied a similar strategy of MTS modification in an inactivation-suppressed mutant (C212S) of ClC-0. Mutation of lysine 165 to cysteine (K165C) rendered the channel nonfunctional, but modification of the introduced cysteine by 2-aminoethyl MTS (MTSEA) recovered functional channels with altered properties of gating-permeation coupling. The fast gate of the MTSEA-modified K165C homodimer responded to external Cl(-) less effectively, so the P(o)-V curve was shifted to a more depolarized potential by approximately 45 mV. The K165C-K165 heterodimer showed double-barrel-like channel activity after MTSEA modification, with the fast-gating behaviors mimicking a combination of those of the mutant and the wild-type pore, as expected for the two-pore model. Without MTSEA modification, the heterodimer showed only one pore, and was easier to inactivate than the two-pore channel. These results showed that K165 is important for both the fast and slow gating of ClC-0. Therefore, the effects of MTS reagents on channel gating need to be carefully considered when interpreting the apparent modification rate.     

Inhibition of ClC-2 Chloride Channels by a Peptide Component or Components of Scorpion Venom

ClC chloride channels play essential roles in membrane excitability and maintenance of osmotic balance. Despite the recent crystallization of two bacterial ClC-like proteins, the gating mechanism for these channels remains unclear. In this study we tested scorpion venom for the presence of novel peptide inhibitors of ClC channels, which might be useful tools for dissecting the mechanisms underlying ClC channel gating. Recently, it has been shown that a peptide component of venom from the scorpion L. quinquestriatus hebraeus inhibits the CFTR chloride channel from the intracellular side. Using two-electrode voltage clamp we studied the effect of scorpion venom on ClC-0, -1, and -2, and found both dose- and voltage-dependent inhibition only of ClC-2. Comparison of voltage-dependence of inhibition by venom to that of known pore blockers revealed opposite voltage dependencies, suggesting different mechanisms of inhibition. Kinetic data show that venom induced slower activation kinetics compared to pre-venom records, suggesting that the active component(s) of venom may function as a gating modifier at ClC-2. Trypsinization abolished the inhibitory activity of venom, suggesting that the component(s) of scorpion venom that inhibits ClC-2 is a peptide.     

Involvement of helices at the dimer interface in ClC-1 common gating

ClC-1 is a dimeric, double-pored chloride channel that is present in skeletal muscle. Mutations of this channel can result in the condition myotonia, a muscle disorder involving increased muscle stiffness. It has been shown that the dominant form of myotonia often results from mutations that affect the so-called slow, or common, gating process of the ClC-1 channel. Mutations causing dominant myotonia are seen to cluster at the interface of the ClC-1 channel monomers. This study has investigated the role of the H, I, P, and Q helices, which lie on this interface, as well as the G helix, which is situated immediately behind the H and I helices, on ClC-1 gating. 11 mutant ClC-1 channels (T268M, C277S, C278S, S289A, T310M, S312A, V321S, T539A, S541A, M559T, and S572V) were produced using site-directed mutagenesis, and gating properties of these channels were investigated using electrophysiological techniques. Six of the seven mutations in G, H, and I, and two of the four mutations in P and Q, caused shifts of the ClC-1 open probability. In the majority of cases this was due to alterations in the common gating process, with only three of the mutants displaying any change in fast gating. Many of the mutant channels also showed alterations in the kinetics of the common gating process, particularly at positive potentials. The changes observed in common gating were caused by changes in the opening rate (e.g. T310M), the closing rate (e.g. C277S), or both rates. These results indicate that mutations in the helices forming the dimer interface are able to alter the ClC-1 common gating process by changing the energy of the open and/or closed channel states, and hence altering transition rates between these states.     

The role of a conserved lysine in chloride- and voltage-dependent ClC-0 fast gating

ClC-0 is a chloride channel whose gating is sensitive to voltage, chloride, and pH. In a previous publication, we showed that the K149C mutation causes a +70-mV shift in the voltage dependence of ClC-0 fast gating. In this paper we analyze the effects of a series of mutations at K149 on the voltage and chloride dependence of gating. By fitting our data to the previously proposed four-state model for ClC-0 fast gating, we show which steps in fast-gate opening are likely to be affected by these mutations. Computational analysis of mutant ClC-0 homology models show electrostatic contributions to chloride binding that may partially account for the effects of K149 on gating. The analysis of gating kinetics in combination with the available structural information suggests some of the structural changes likely to underpin fast-gate opening.     

Mechanism of inverted activation of ClC-1 channels caused by a novel myotonia congenita mutation

The voltage-gated chloride channel ClC-1 is the major contributor of membrane conductance in skeletal muscle and has been associated with the inherited muscular disorder myotonia congenita. Here, we report a novel mutation identified in a recessive myotonia congenita family. This mutation, Gly-499 to Arg (G499R) is located in the putative transmembrane domain 10 of the ClC-1 protein. In contrast to normal ClC-1 channels that deactivate upon hyperpolarization, functional expression of G499R ClC-1 yielded a hyperpolarization-activated chloride current when measured in the presence of a high (134 mM) intracellular chloride concentration. Current was abolished when measured with a physiological chloride transmembrane gradient. Electrophysiological analysis of other Gly-499 mutants (G499K, G499Q, and G499E) suggests that the positive charge introduced by the G499R mutation may be responsible for this unique gating behavior. To further explore the function of domain 10, we mutated two charged residues near Gly-499 of ClC-1. Functional analyses of R496Q, R496Q/G499R, R496K, and E500Q mutant channels suggest that the charged residues in domain 10 are important for normal channel function. Study of these mutants may shed further light on the structure and voltage-gating of this channel.     

Quantitative analysis of the voltage-dependent gating of mouse parotid ClC-2 chloride channel

Various ClC-type voltage-gated chloride channel isoforms display a double barrel topology, and their gating mechanisms are thought to be similar. However, we demonstrate in this work that the nearly ubiquitous ClC-2 shows significant differences in gating when compared with ClC-0 and ClC-1. To delineate the gating of ClC-2 in quantitative terms, we have determined the voltage (V(m)) and time dependence of the protopore (P(f)) and common (P(s)) gates that control the opening and closing of the double barrel. mClC-2 was cloned from mouse salivary glands, expressed in HEK 293 cells, and the resulting chloride currents (I(Cl)) were measured using whole cell patch clamp. WT channels had I(Cl) that showed inward rectification and biexponential time course. Time constants of fast and slow components were approximately 10-fold different at negative V(m) and corresponded to P(f) and P(s), respectively. P(f) and P(s) were approximately 1 at -200 mV, while at V(m) > or = 0 mV, P(f) approximately 0 and P(s) approximately 0.6. Hence, P(f) dominated open kinetics at moderately negative V(m), while at very negative V(m) both gates contributed to gating. At V(m) > or = 0 mV, mClC-2 closes by shutting off P(f). Three- and two-state models described the open-to-closed transitions of P(f) and P(s), respectively. To test these models, we mutated conserved residues that had been previously shown to eliminate or alter P(f) or P(s) in other ClC channels. Based on the time and V(m) dependence of the two gates in WT and mutant channels, we constructed a model to explain the gating of mClC-2. In this model the E213 residue contributes to P(f), the dominant regulator of gating, while the C258 residue alters the V(m) dependence of P(f), probably by interacting with residue E213. These data provide a new perspective on ClC-2 gating, suggesting that the protopore gate contributes to both fast and slow gating and that gating relies strongly on the E213 residue.     

The chloride channel ClC-4 co-localizes with cystic fibrosis transmembrane conductance regulator and may mediate chloride flux across the apical membrane of intestinal epithelia.

Cystic fibrosis (CF) causing mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) lead to mislocalization of CFTR protein from the brush border membrane of epithelial tissues and/or its dysfunction as a chloride channel. In initial reports, it was proposed that certain channels from the ClC family of chloride channels may provide compensatory or alternative pathways for epithelial chloride secretion in tissues from cystic fibrosis patients. In the present work, we provide the first evidence that ClC-4 protein is functionally expressed on the surface of the intestinal epithelium and hence, is appropriately localized to act as a therapeutic target in this CF-affected tissue. We show using confocal and electron microscopy that ClC-4 co-localizes with CFTR in the brush border membrane of the epithelium lining intestinal crypts in mouse and human tissues. In Caco-2 cells, a cell line thought to model human enterocytes, ClC-4 protein is expressed on the cell surface and also partially co-localizes with EEA1 and transferrin, marker molecules of early and recycling endosomes, respectively. Hence, like CFTR, ClC-4 may cycle between the plasma membrane and endosomal compartment. Furthermore, we show that ClC-4 functions as a chloride channel on the surface of these epithelial cells as antisense ClC-4 cDNA expression reduced the amplitude of endogenous chloride currents by 50%. These studies provide the first evidence that ClC-4 is endogenously expressed and may be functional in the brush border membrane of enterocytes and hence should be considered as a candidate channel to provide an alternative pathway for chloride secretion in the gastrointestinal tract of CF patients.     

The mechanism of fast-gate opening in ClC-0

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.     

Cysteine accessibility in ClC-0 supports conservation of the ClC intracellular vestibule

ClC chloride channels, which are ubiquitously expressed in mammals, have a unique double-barreled structure, in which each monomer forms its own pore. Identification of pore-lining elements is important for understanding the conduction properties and unusual gating mechanisms of these channels. Structures of prokaryotic ClC transporters do not show an open pore, and so may not accurately represent the open state of the eukaryotic ClC channels. In this study we used cysteine-scanning mutagenesis and modification (SCAM) to screen >50 residues in the intracellular vestibule of ClC-0. We identified 14 positions sensitive to the negatively charged thiol-modifying reagents sodium (2-sulfonatoethyl)methanethiosulfonate (MTSES) or sodium 4-acetamido-4'-maleimidylstilbene-2'2-disulfonic acid (AMS) and show that 11 of these alter pore properties when modified. In addition, two MTSES-sensitive residues, on different helices and in close proximity in the prokaryotic structures, can form a disulfide bond in ClC-0. When mapped onto prokaryotic structures, MTSES/AMS-sensitive residues cluster around bound chloride ions, and the correlation is even stronger in the ClC-0 homology model developed by Corry et al. (2004). These results support the hypothesis that both secondary and tertiary structures in the intracellular vestibule are conserved among ClC family members, even in regions of very low sequence similarity.     

A single conductance pore for chloride ions formed by two cystic fibrosis transmembrane conductance regulator molecules

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-dependent protein kinase (PKA)- and ATP-regulated chloride channel, whose gating process involves intra- or intermolecular interactions among the cytosolic domains of the CFTR protein. Tandem linkage of two CFTR molecules produces a functional chloride channel with properties that are similar to those of the native CFTR channel, including trafficking to the plasma membrane, ATP- and PKA-dependent gating, and a unitary conductance of 8 picosiemens (pS). A heterodimer, consisting of a wild type and a mutant CFTR, also forms an 8-pS chloride channel with mixed gating properties of the wild type and mutant CFTR channels. The data suggest that two CFTR molecules interact together to form a single conductance pore for chloride ions.     

Conduction mechanisms of chloride ions in ClC-type channels

The conduction properties of ClC-0 and ClC-1 chloride channels are examined using electrostatic calculations and three-dimensional Brownian dynamics simulations. We create an open-state configuration of the prokaryotic ClC Cl(-) channel using its known crystallographic structure as a basis. Two residues that are occluding the channel are slowly pushed outward with molecular dynamics to create a continuous ion-conducting path with the minimum radius of 2.5 A. Then, retaining the same pore shape, the prokaryotic ClC channel is converted to either ClC-0 or ClC-1 by replacing all the nonconserved dipole-containing and charged amino acid residues. Employing open-state ClC-0 and ClC-1 channel models, current-voltage curves consistent with experimental measurements are obtained. We find that conduction in these pores involves three ions. We locate the binding sites, as well as pinpointing the rate-limiting steps in conduction, and make testable predictions about how the single channel current across ClC-0 and ClC-1 will vary as the ionic concentrations are increased. Finally, we demonstrate that a ClC-0 homology model created from an alternative sequence alignment fails to replicate any of the experimental observations.     

Oxidation and reduction control of the inactivation gating of Torpedo ClC-0 chloride channels

Oxidation and reduction (redox) are known to modulate the function of a variety of ion channels. Here, we report a redox regulation of the function of ClC-0, a chloride (Cl(-)) channel from the Torpedo electric organ. The study was motivated by the occasional observation of oocytes with hyperpolarization-activated Cl(-) current when these oocytes expressed ClC-0. We find that these atypical recording traces can be turned into typical ClC-0 current by incubating the oocyte in millimolar concentrations of reducing agents, suggesting that the channel function is regulated by oxidation and reduction. The redox control apparently results from an effect of oxidation on the slow (inactivation) gating: oxidation renders it more difficult for the channel to recover from the inactivated states. Introducing the point mutation C212S in ClC-0 suppresses the inactivation state, and this inactivation-suppressed mutant is no longer sensitive to the inhibition by oxidizing reagents. However, C212 is probably not the target for the redox reaction because the regulation of the inactivation gating by oxidation is still present in a pore mutant (K165C/K165 heterodimer) in which the C212S mutation is present. Taking advantage of the K165C/K165 heterodimer, we further explore the oxidation effect in ClC-0 by methane thiosulfonate (MTS) modifications. We found that trimethylethylammonium MTS modification of the introduced cysteine can induce current in the K165C/K165 heterodimer, an effect attributed to the recovery of the channel from the inactivation state. The current induction by MTS reagents is subjected to redox controls, and thus the extent of this current induction can serve as an indicator to report the oxidation state of the channel. These results together suggest that the inactivation gating of ClC-0 is affected by redox regulation. The finding also provides a convenient method to "cure" those atypical recording traces of ClC-0 expressed in Xenopus oocytes.     

Single-channel analysis of a ClC-2-like chloride conductance in cultured rat cortical astrocytes

The single-channel behavior of the hyperpolarization-activated, ClC-2-like inwardly rectifying Cl- current (IClh), induced by long-term dibutyryl-cyclic-AMP-treated cultured cortical rat astrocytes, was analyzed with the patch-clamp technique. In outside-out patches in symmetrical 144 mM Cl-solutions, openings of hyperpolarization-activated small-conductance Cl channels revealed burst activity of two equidistant conductance levels of 3 and 6 pS. The unitary openings displayed slow activation kinetics. The probabilities of the closed and conducting states were consistent with a double-barrelled structure of the channel protein. These results suggest that the astrocytic ClC-2-like Cl- current Iclh is mediated by a small-conductance Cl channel, which has the same structural motif as the Cl- channel prototype CIC-0.     

Anion permeation in human ClC-4 channels

ClC-4 and ClC-5 are mammalian ClC isoforms with unique ion conduction and gating properties. Macroscopic current recordings in heterologous expression systems revealed very small currents at negative potentials, whereas a substantially larger instantaneous current amplitude and a subsequent activation were observed upon depolarization. Neither the functional basis nor the physiological impact of these channel features are currently understood. Here, we used whole-cell recordings to study pore properties of human ClC-4 channels heterologously expressed in tsA201 or HEK293 cells. Variance analysis demonstrated that the prominent rectification of the instantaneous macroscopic current amplitude is due to a voltage-dependent unitary current conductance. The single channel amplitudes are very small, i.e., 0.10 +/- 0.02 pA at +140 mV for external Cl(-) and internal I(-). Conductivity and permeability sequences were determined for various external and internal anions, and both values increase for anions with lower dehydration energies. ClC-4 exhibits pore properties that are distinct from other ClC isoforms. These differences can be explained by assuming differences in the size of the pore narrowing and the electrostatic potentials within the ion conduction pathways.     

Roles of K149, G352, and H401 in the channel functions of ClC-0: testing the predictions from theoretical calculations

The ClC family of Cl(-) channels and transporters comprises membrane proteins ubiquitously present in species ranging from prokaryotes to mammals. The recently solved structures of the bacterial ClC proteins have provided a good model to guide the functional experiments for the eukaryotic Cl(-) channels. Theoretical calculations based on the bacterial ClC structures have identified several residues critical for the Cl(-) binding energy in the Cl(-) transport pathway. It was speculated that the corresponding residues in eukaryotic Cl(-) channels might play similar roles for the channel functions. In this study, we made a series of mutations in three such residues in eukaryotic ClC Cl(-) channels (K149, G352, and H401 in ClC-0) and studied the functional consequences on the channel properties. A cysteine modification approach was also employed to evaluate the electrostatic effects of the charge placed at these three positions. The experimental results revealed that among the three residues tested, K149 plays the most important role in controlling both the gating and the permeation functions of ClC-0. On the other hand, mutations of H401 alter the channel conductance but not the gating properties, while mutations of G352 result in very little functional consequence. The mutation of K149 into a neutral residue leucine (K149L) shifts the activation curve and leads to flickery channel openings. The anion permeability ratios derived from bi-ionic experiments are also significantly altered in that the selectivity of Cl(-) over other anions is decreased. Furthermore, removing the positive charge at this position reduces and increases, respectively, the accessibility of the negatively and positively charged methane thiosulfonate reagents to the pore. The control of the accessibility to charged MTS reagents and the regulation of the anion permeation support the idea that K149 exerts an electrostatic effect on the channel function, confirming the prediction from computational studies.     

A synthetic prostone activates apical chloride channels in A6 epithelial cells

The bicyclic fatty acid lubiprostone (formerly known as SPI-0211) activates two types of anion channels in A6 cells. Both channel types are rarely, if ever, observed in untreated cells. The first channel type was activated at low concentrations of lubiprostone (<100 nM) in >80% of cell-attached patches and had a unit conductance of approximately 3-4 pS. The second channel type required higher concentrations (>100 nM) of lubiprostone to activate, was observed in approximately 30% of patches, and had a unit conductance of 8-9 pS. The properties of the first type of channel were consistent with ClC-2 and the second with CFTR. ClC-2's unit current strongly inwardly rectified that could be best fit by models of the channel with multiple energy barrier and multiple anion binding sites in the conductance pore. The open probability and mean open time of ClC-2 was voltage dependent, decreasing dramatically as the patches were depolarized. The order of anion selectivity for ClC-2 was Cl > Br > NO(3) > I > SCN, where SCN is thiocyanate. ClC-2 was a "double-barreled" channel favoring even numbers of levels over odd numbers as if the channel protein had two conductance pathways that opened independently of one another. The channel could be, at least, partially blocked by glibenclamide. The properties of the channel in A6 cells were indistinguishable from ClC-2 channels stably transfected in HEK293 cells. CFTR in the patches had a selectivity of Cl > Br > NO(3) congruent with SCN congruent with I. It outwardly rectified as expected for a single-site anion channel. Because of its properties, ClC-2 is uniquely suitable to promote anion secretion with little anion reabsorption. CFTR, on the other hand, could promote either reabsorption or secretion depending on the anion driving forces.