Anion- and proton-dependent gating of ClC-4 anion/proton transporter under uncoupling conditions

ClC-4 is a secondary active transporter that exchanges Cl⁻ ions and H⁺ with a 2:1 stoichiometry. In external SCN⁻, ClC-4 becomes uncoupled and transports anions with high unitary transport rate. Upon voltage steps, the number of active transporters varies in a time-dependent manner, resembling voltage-dependent gating of ion channels. We here investigated modification of the voltage dependence of uncoupled ClC-4 by protons and anions to quantify association of substrates with the transporter. External acidification shifts voltage dependence of ClC-4 transport to more positive potentials and leads to reduced transport currents. Internal pH changes had less pronounced effects. Uncoupled ClC-4 transport is facilitated by elevated external [SCN⁻] but impaired by internal Cl⁻ and I⁻. Block by internal anions indicates the existence of an internal anion-binding site with high affinity that is not present in ClC channels. The voltage dependence of ClC-4 coupled transport is modulated by external protons and internal Cl⁻ in a manner similar to what is observed under uncoupling conditions. Our data illustrate functional differences but also similarities between ClC channels and transporters.     

Glutamate 268 regulates transport probability of the anion/proton exchanger ClC-5

The Cl(-)/H(+) exchange mediated by ClC transporters can be uncoupled by external SCN(-) and mutations of the proton glutamate, a conserved residue at the internal side of the protein. We show here for the mammalian ClC transporter ClC-5 that acidic internal pH led to a greater increase in currents upon exchanging extracellular Cl(-) for SCN(-). However, transport uncoupling, unitary current amplitudes, and the voltage dependence of the depolarization-induced activation were not altered by low pH values. Therefore, it is likely that an additional gating process regulates ClC-5 transport. Higher internal [H(+)] and the proton glutamate mutant E268H altered the ratio between ClC-5 transport and nonlinear capacitance, indicating that the gating charge movements in ClC-5 arise from incomplete transport cycles and that internal protons increase the transport probability of ClC-5. This was substantiated by site-directed sulfhydryl modification of the proton glutamate mutant E268C. The mutation exhibited small transport currents together with prominent gating charge movements. The charge restoration using a negatively charged sulfhydryl reagent reinstated also the WT phenotype. Neutralization of the charge of the gating glutamate 211 by the E211C mutation abolished the effect of internal protons, showing that the increased transport probability of ClC-5 results from protonation of this residue. S168P (a mutation that decreases the anion affinity of the central binding site) reduced also the internal pH dependence of ClC-5. These results support the idea that protonation of the gating glutamate 211 at the central anion-binding site of ClC-5 is mediated by the proton glutamate 268.     

Carboxy-terminal truncations modify the outer pore vestibule of muscle chloride channels

Mammalian ClC-type chloride channels have large cytoplasmic carboxy-terminal domains whose function is still insufficiently understood. We investigated the role of the distal part of the carboxy-terminus of the muscle isoform ClC-1 by constructing and functionally evaluating two truncation mutants, R894X and K875X. Truncated channels exhibit normal unitary conductances and anion selectivities but altered apparent anion binding affinities in the open and in the closed state. Since voltage-dependent gating is strictly coupled to ion permeation in ClC-1 channels, the changed pore properties result in different fast and slow gating. Full length and truncated channels also differed in methanethiosulphonate (MTS) modification rate constants of an engineered cysteine at position 231 near the selectivity filter. Our data demonstrate that the carboxy-terminus of ClC channels modifies the conformation of the outer pore vestibule.     

Overexpression of CLC-3 in HEK293T cells yields novel currents that are pH dependent

ClC-3 is a member of the ClC family of anion channels/transporters. Recently, the closely related proteins ClC-4 and ClC-5 were shown to be Cl(-)/H(+) antiporters (39, 44). The function of ClC-3 has been controversial. We studied anion currents in HEK293T cells expressing wild-type or mutant ClC-3. The basic biophysical properties of ClC-3 currents were very similar to those of ClC-4 and ClC-5, and distinct from those of the swelling-activated anion channel. ClC-3 expression induced currents with time-dependent activation that rectified sharply in the outward direction. The reversal potential of the current shifted by -48.3 +/- 2.5 mV per 10-fold (decade) change in extracellular Cl(-) concentration, which did not conform to the behavior of an anion-selective channel based upon the Nernst equation, which predicts a -58.4 mV/decade shift at 22 degrees C. Manipulation of extracellular pH (6.35-8.2) altered reversal potential by 10.2 +/- 3.0 mV/decade, suggesting that ClC-3 currents were coupled to proton movement. Mutation of a specific glutamate residue (E224A) changed voltage dependence in a manner similar to that observed in other ClC Cl(-)/H(+) antiporters. Mutant currents exhibited Nernstian changes in reversal potential in response to altered extracellular Cl(-) concentration that averaged -60 +/- 3.4 mV/decade and were pH independent. Thus ClC-3 overexpression induced a pH-sensitive conductance in HEK293T cells that is biophysically similar to ClC-4 and ClC-5.     

Ion-binding properties of the ClC chloride selectivity filter

The ClC channels are members of a large protein family of chloride (Cl-) channels and secondary active Cl- transporters. Despite their diverse functions, the transmembrane architecture within the family is conserved. Here we present a crystallographic study on the ion-binding properties of the ClC selectivity filter in the close homolog from Escherichia coli (EcClC). The ClC selectivity filter contains three ion-binding sites that bridge the extra- and intracellular solutions. The sites bind Cl- ions with mM affinity. Despite their close proximity within the filter, the three sites can be occupied simultaneously. The ion-binding properties are found conserved from the bacterial transporter EcClC to the human Cl- channel ClC-1, suggesting a close functional link between ion permeation in the channels and active transport in the transporters. In resemblance to K+ channels, ions permeate the ClC channel in a single file, with mutual repulsion between the ions fostering rapid conduction.     

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.     

The Late Endosomal ClC-6 Mediates Proton/Chloride Countertransport in Heterologous Plasma Membrane Expression

Members of the CLC protein family of Cl⁻ channels and transporters display the remarkable ability to function as either chloride channels or Cl⁻/H⁺ antiporters. Due to the intracellular localization of ClC-6 and ClC-7, it has not yet been possible to study the biophysical properties of these members of the late endosomal/lysosomal CLC branch in heterologous expression. Whereas recent data suggest that ClC-7 functions as an antiporter, transport characteristics of ClC-6 have remained entirely unknown. Here, we report that fusing the green fluorescent protein (GFP) to the N terminus of ClC-6 increased its cell surface expression, allowing us to functionally characterize ClC-6. Compatible with ClC-6 mediating Cl⁻/H⁺ exchange, Xenopus oocytes expressing GFP-tagged ClC-6 alkalinized upon depolarization. This alkalinization was dependent on the presence of extracellular anions and could occur against an electrochemical proton gradient. As observed in other CLC exchangers, ClC-6-mediated H⁺ transport was abolished by mutations in either the “gating” or “proton” glutamate. Overexpression of GFP-tagged ClC-6 in CHO cells elicited small, outwardly rectifying currents with a Cl⁻ > I⁻ conductance sequence. Mutating the gating glutamate of ClC-6 yielded an ohmic anion conductance that was increased by additionally mutating the “anion-coordinating” tyrosine. Additionally changing the chloride-coordinating serine 157 to proline increased the NO₃⁻ conductance of this mutant. Taken together, these data demonstrate for the first time that ClC-6 is a Cl⁻/H⁺ antiporter.     

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.     

Determinants of anion-proton coupling in mammalian endosomal CLC proteins

Many proteins of the CLC gene family are Cl(-) channels, whereas others, like the bacterial ecClC-1 or mammalian ClC-4 and -5, mediate Cl(-)/H(+) exchange. Mutating a "gating glutamate" (Glu-224 in ClC-4 and Glu-211 in ClC-5) converted these exchangers into anion conductances, as did the neutralization of another, intracellular "proton glutamate" in ecClC-1. We show here that neutralizing the proton glutamate of ClC-4 (Glu-281) and ClC-5 (Glu-268), but not replacing it with aspartate, histidine, or tyrosine, rather abolished Cl(-) and H(+) transport. Surface expression was unchanged by these mutations. Uncoupled Cl(-) transport could be restored in the ClC-4(E281A) and ClC-5(E268A) proton glutamate mutations by additionally neutralizing the gating glutamates, suggesting that wild type proteins transport anions only when protons are supplied through a cytoplasmic H(+) donor. Each monomeric unit of the dimeric protein was found to be able to carry out Cl(-)/H(+) exchange independently from the transport activity of the neighboring subunit. NO(3)(-) or SCN(-) transport was partially uncoupled from H(+) countertransport but still depended on the proton glutamate. Inserting proton glutamates into CLC channels altered their gating but failed to convert them into Cl(-)/H(+) exchangers. Noise analysis indicated that ClC-5 switches between silent and transporting states with an apparent unitary conductance of 0.5 picosiemens. Our results are consistent with the idea that Cl(-)/H(+) exchange of the endosomal ClC-4 and -5 proteins relies on proton delivery from an intracellular titratable residue at position 268 (numbering of ClC-5) and that the strong rectification of currents arises from the voltage-dependent proton transfer from Glu-268 to Glu-211.     

A Conserved Aspartate Determines Pore Properties of Anion Channels Associated with Excitatory Amino Acid Transporter 4 (EAAT4)

Excitatory amino acid transporter (EAAT) glutamate transporters function not only as secondary active glutamate transporters but also as anion channels. Recently, a conserved aspartic acid (Asp¹¹²) within the intracellular loop near to the end of transmembrane domain 2 was proposed as a major determinant of substrate-dependent gating of the anion channel associated with the glial glutamate transporter EAAT1. We studied the corresponding mutation (D117A) in another EAAT isoform, EAAT4, using heterologous expression in mammalian cells, whole cell patch clamp, and noise analysis. In EAAT4, D117A modifies unitary conductances, relative anion permeabilities, as well as gating of associated anion channels. EAAT4 anion channel gating is characterized by two voltage-dependent gating processes with inverse voltage dependence. In wild type EAAT4, external l-glutamate modifies the voltage dependence as well as the minimum open probabilities of both gates, resulting in concentration-dependent changes of the number of open channels. Not only transport substrates but also anions affect wild type EAAT4 channel gating. External anions increase the open probability and slow down relaxation constants of one gating process that is activated by depolarization. D117A abolishes the anion and glutamate dependence of EAAT4 anion currents and shifts the voltage dependence of EAAT4 anion channel activation by more than 200 mV to more positive potentials. D117A is the first reported mutation that changes the unitary conductance of an EAAT anion channel. The finding that mutating a pore-forming residue modifies gating illustrates the close linkage between pore conformation and voltage- and substrate-dependent gating in EAAT4 anion channels.     

Functional characterization of a ClC transporter by solid-supported membrane electrophysiology

EcClC, a prokaryotic member of the ClC family of chloride channels and transporters, works as coupled H⁺/Cl⁻ exchanger. With a known structure and the possibility of investigating its behavior with different biochemical and biophysical techniques, the protein has become an important model system for the family. Although many aspects of its function have been previously characterized, it was difficult to measure transport on the same sample under different environmental conditions. To overcome this experimental limitation, we have studied EcClC by solid-supported membrane electrophysiology. The large transport-related transient currents and a simple way of relating transport rates to the measured signal have allowed a thorough investigation of ion selectivity, inhibition, and the dependence of transport on changes in ion concentration and pH. Our results confirm that the protein transports larger anions with about similar rates, whereas the smaller fluoride is not a substrate. We also show that 4,4′-diisothiocyano-2,2’-stilbenedisulfonic acid (DIDS), a known inhibitor of other anion transport protein, irreversibly inhibits EcClC from the intracellular side. The chloride dependence shows an apparent saturation at millimolar concentrations that resembles a similar behavior in eukaryotic ClC channels. Our experiments have also allowed us to quantify the pH dependence of transport. EcClC shows a strong activation at low pH with an apparent pKa of 4.6. The pronounced pH dependence is lost by the mutation of a conserved glutamate facing the extracellular solution that was previously shown to be an acceptor for transported protons, whereas it is largely retained by the mutation of an equivalent residue at the intracellular side. Our results have provided a quantitative basis for the transport behavior of EcClC, and they will serve as a reference for future investigations of novel electrogenic transporters with still-uncharacterized properties.     

A cation counterflux supports lysosomal acidification

The profound luminal acidification essential for the degradative function of lysosomes requires a counter-ion flux to dissipate an opposing voltage that would prohibit proton accumulation. It has generally been assumed that a parallel anion influx is the main or only counter-ion transport that enables acidification. Indeed, defective anion conductance has been suggested as the mechanism underlying attenuated lysosome acidification in cells deficient in CFTR or ClC-7. To assess the individual contribution of counter-ions to acidification, we devised means of reversibly and separately permeabilizing the plasma and lysosomal membranes to dialyze the cytosol and lysosome lumen in intact cells, while ratiometrically monitoring lysosomal pH. Replacement of cytosolic Cl⁻ with impermeant anions did not significantly alter proton pumping, while the presence of permeant cations in the lysosomal lumen supported acidification. Accordingly, the lysosomes were found to acidify to the same pH in both CFTR- and ClC-7–deficient cells. We conclude that cations, in addition to chloride, can support lysosomal acidification and defects in lysosomal anion conductance cannot explain the impaired microbicidal capacity of CF phagocytes.     

The structure of the cytoplasmic domain of the chloride channel ClC-Ka reveals a conserved interaction interface

The cytoplasmic domains of ClC chloride channels and transporters are ubiquitously found in eukaryotic family members and have been suggested to be involved in the regulation of ion transport. All cytoplasmic ClC domains share a conserved scaffold that contains a pair of CBS motifs. Here we describe the structure of the cytoplasmic component of the human chloride channel ClC-Ka at 1.6 A resolution. The structure reveals a dimeric organization of the domain that is unusual for CBS motif containing proteins. Using a biochemical approach combining mutagenesis, crosslinking, and analytical ultracentrifugation, we demonstrate that the interaction interface is preserved in solution and that the distantly related channel ClC-0 likely exhibits a similar structural organization. Our results reveal a conserved interaction interface that relates the cytoplasmic domains of ClC proteins and establish a structural relationship that is likely general for this important family of transport proteins.     

Early cell death induced by Clostridium difficile TcdB: Uptake and Rac1‐glucosylation kinetics are decisive for cell fate

Toxin A and Toxin B (TcdA/TcdB) are large glucosyltransferases produced by Clostridium difficile. TcdB but not TcdA induces reactive oxygen species‐mediated early cell death (ECD) when applied at high concentrations. We found that nonglucosylated Rac1 is essential for induction of ECD since inhibition of Rac1 impedes this effect. ECD only occurs when TcdB is rapidly endocytosed. This was shown by generation of chimeras using the trunk of TcdB from a hypervirulent strain. TcdB from hypervirulent strain has been described to translocate from endosomes at higher pH values and thus, meaning faster than reference type TcdB. Accordingly, intracellular delivery of the glucosyltransferase domain of reference TcdB by the trunk of TcdB from hypervirulent strain increased ECD. Furthermore, proton transporters such as sodium/proton exchanger (NHE) or the ClC‐5 anion/proton exchanger, both of which contribute to endosomal acidification, also affected cytotoxic potency of TcdB: Specific inhibition of NHE reduced cytotoxicity, whereas transfection of cells with the endosomal anion/proton exchanger ClC‐5 increased cytotoxicity of TcdB. Our data suggest that both the uptake rate of TcdB into the cytosol and the status of nonglucosylated Rac1 are key determinants that are decisive for whether ECD or delayed apoptosis is triggered.     

Isolation and Characterization of a High Affinity Peptide Inhibitor of ClC-2 Chloride Channels

The ClC protein family includes voltage-gated chloride channels and chloride/proton exchangers. In eukaryotes, ClC proteins regulate membrane potential of excitable cells, contribute to epithelial transport, and aid in lysosomal acidification. Although structure/function studies of ClC proteins have been aided greatly by the available crystal structures of a bacterial ClC chloride/proton exchanger, the availability of useful pharmacological tools, such as peptide toxin inhibitors, has lagged far behind that of their cation channel counterparts. Here we report the isolation, from Leiurus quinquestriatus hebraeus venom, of a peptide toxin inhibitor of the ClC-2 chloride channel. This toxin, GaTx2, inhibits ClC-2 channels with a voltage-dependent apparent K_D of ∼20 pm, making it the highest affinity inhibitor of any chloride channel. GaTx2 slows ClC-2 activation by increasing the latency to first opening by nearly 8-fold but is unable to inhibit open channels, suggesting that this toxin inhibits channel activation gating. Finally, GaTx2 specifically inhibits ClC-2 channels, showing no inhibitory effect on a battery of other major classes of chloride channels and voltage-gated potassium channels. GaTx2 is the first peptide toxin inhibitor of any ClC protein. The high affinity and specificity displayed by this toxin will make it a very powerful pharmacological tool to probe ClC-2 structure/function.ClC proteins form a family of voltage-gated Cl⁻ channels and Cl⁻/H⁺ exchangers that are found in animals, plants, and bacteria (1). These proteins are expressed on the plasma membrane and some intracellular membranes in both excitable and nonexcitable cells (1, 2). There are nine mammalian members of the ClC family that perform functions as varied as maintenance of membrane potential in neuronal cells (ClC-2) (3), Cl⁻ transport across plasma membranes of epithelial and skeletal muscle cells (ClC-1, ClC-2, and ClC-Ka/b) (1, 4), and participation in lysosomal acidification (ClC-5 and ClC-6) (2). Defects in the genes encoding ClC proteins are linked to a number of diseases including myotonia, epilepsy, Dent''s disease, and Bartter''s syndrome (1–3). It has been suggested recently that ClC-2 may play a role in constipation-associated irritable bowel disease as well as in atherosclerosis (5, 6). Most ClC channels show localized tissue expression; ClC-1, for example, is expressed solely in skeletal muscle, whereas ClC-Ka/b is localized to the kidney. ClC-2, on the other hand, is expressed nearly ubiquitously, suggesting that this channel plays an important, yet largely undefined, physiological role (1, 2).ClC proteins are structurally unrelated to cation channels, with the functional unit being a homodimer (1). ClC channels display two equidistant conductance levels for a single channel opening. In 2002, the crystal structure of a bacterial ClC protein from Salmonella typhimurium was solved, revealing a very complicated membrane topology consisting of 18 α-helical units/subunit in the homodimer, only some of which fully traverse the membrane (7). Examination of the crystal structure revealed no obvious pore, such as is evident in K⁺ channel structures, even though bound Cl⁻ ions were present near the proposed selectivity filter (7, 8). Shortly after the crystal structure was solved, it was shown that the bacterial ClC protein was actually a Cl⁻/H⁺ exchanger and not a channel (9). Comparison of the amino acid sequence of the bacterial ClC protein with that of the eukaryotic ClC channels ClC-0, -1, and -2 revealed only 22, 16, and 19% overall identity, respectively (data not shown). The divergence is largely in the cytoplasmic domains, which are absent in bacterial ClC proteins; sequence identity is much higher in the transmembrane domains.Single-channel gating in ClC proteins is complicated, involving both fast and slow gating processes, which are thought to involve separate regions of the protein (1). Fast gating controls the opening and closing of both protopores independently, operating on the millisecond time scale or faster. Through examination of the crystal structure and subsequent electrophysiological analysis, the fast gating process was revealed to involve a conserved glutamate residue deep within each pore (10). This acidic residue lies near a Cl⁻-binding site and moves slightly to open the pathway in response to changes in membrane voltage and subsequent changes in occupancy of that site, thus providing the link between permeation and gating observed in ClC channels (4). In contrast, slow gating controls both pores simultaneously, operating on the hundreds of milliseconds to seconds time scale. Unlike with fast gating, the regions of the ClC protein involved in slow gating are still unknown, despite the availability of the bacterial ClC crystal structure. It is believed that the dimer interface contributes to slow gating, as well as the long cytoplasmic C-terminal domain, an isolated version of which was recently crystallized (11–13). However, the conformational changes involved in the fast and slow gating processes are still largely unknown. Also, in both ClC-1 and -2, fast and slow gating are linked through an undetermined mechanism (14, 15).Despite the availability of the bacterial ClC protein crystal structure, our understanding of gating mechanisms and structural rearrangements of ClC proteins has lagged behind that of their cation channel counterparts. This is due in large part to a lack of useful pharmacological agents, such as peptide toxins, that may be used as tools. Toxins from venomous animals such as scorpions, snakes, and cone snails have been used for a number of years to define the permeation pathways and gating processes of cation channels (16). However, no peptide toxins have been isolated that inhibit a ClC channel, and only one toxin has been isolated that inhibits any Cl⁻ channel of known molecular identity (17). We recently showed that venom from the scorpion Leiurus quinquestriatus hebraeus contains a peptide component that inhibits the ClC-2 chloride channel (18). Here, we report the isolation of this peptide toxin, its proteomic properties, and primary characteristics of the biophysical mechanism of inhibition.     

High-level expression, functional reconstitution, and quaternary structure of a prokaryotic ClC-type chloride channel.

ClC-type anion-selective channels are widespread throughout eukaryotic organisms. BLAST homology searches reveal that many microbial genomes also contain members of the ClC family. An Escherichia coli-derived ClC Cl(-) channel homologue, "EriC," the product of the yadQ gene, was overexpressed in E. coli and purified in milligram quantities in a single-step procedure. Reconstitution of purified EriC into liposomes confers on these membranes permeability to anions with selectivity similar to that observed electrophysiologically in mammalian ClC channels. Cross-linking studies argue that EriC is a homodimer in both detergent micelles and reconstituted liposomes, a conclusion corroborated by gel filtration and analytical sedimentation experiments.     

Neuronal ClC-3 Splice Variants Differ in Subcellular Localizations,but Mediate Identical Transport Functions

ClC-3 is a member of the CLC family of anion channels and transporters, for which multiple functional properties and subcellular localizations have been reported. Since alternative splicing often results in proteins with diverse properties, we investigated to what extent alternative splicing might influence subcellular targeting and function of ClC-3. We identified three alternatively spliced ClC-3 isoforms, ClC-3a, ClC-3b, and ClC-3c, in mouse brain, with ClC-3c being the predominant splice variant. Whereas ClC-3a and ClC-3b are present in late endosomes/lysosomes, ClC-3c is targeted to recycling endosomes via a novel N-terminal isoleucine-proline (IP) motif. Surface membrane insertion of a fraction of ClC-3c transporters permitted electrophysiological characterization of this splice variant through whole-cell patch clamping on transfected mammalian cells. In contrast, neutralization of the N-terminal dileucine-like motifs was required for functional analysis of ClC-3a and ClC-3b. Heterologous expression of ClC-3a or ClC-3b carrying mutations in N-terminal dileucine motifs as well as WTClC-3c in HEK293T cells resulted in outwardly rectifying Cl⁻ currents with significant capacitive current components. We conclude that alternative splicing of Clcn3 results in proteins with different subcellular localizations, but leaves the transport function of the proteins unaffected.     

Activation and Proton Transport Mechanism in Influenza A M2 Channel

Molecular dynamics trajectories 2 μs in length have been generated for the pH-activated, tetrameric M2 proton channel of the influenza A virus in all protonation states of the pH sensor located at the His³⁷ tetrad. All simulated structures are in very good agreement with high-resolution structures. Changes in the channel caused by progressive protonation of His³⁷ provide insight into the mechanism of proton transport. The channel is closed at both His³⁷ and Trp⁴¹ sites in the singly and doubly protonated states, but it opens at Trp⁴¹ upon further protonation. Anions access the charged His³⁷ and by doing so stabilize the protonated states of the channel. The narrow opening at the His³⁷ site, further blocked by anions, is inconsistent with the water-wire mechanism of proton transport. Instead, conformational interconversions of His³⁷ correlated with hydrogen bonding to water molecules indicate that these residues shuttle protons in high-protonation states. Hydrogen bonds between charged and uncharged histidines are rare. The valve at Val²⁷ remains on average quite narrow in all protonation states but fluctuates sufficiently to support water and proton transport. A proton transport mechanism in which the channel, depending on pH, opens at either the histidine or valine gate is only partially supported by the simulations.     

Inward Rectification in ClC-0 Chloride Channels Caused by Mutations in Several Protein Regions

Several cloned ClC-type Cl⁻ channels open and close in a voltage-dependent manner. The Torpedo electric organ Cl⁻ channel, ClC-0, is the best studied member of this gene family. ClC-0 is gated by a fast and a slow gating mechanism of opposite voltage direction. Fast gating is dependent on voltage and on the external and internal Cl⁻ concentration, and it has been proposed that the permeant anion serves as the gating charge in ClC-0 (Pusch, M., U. Ludewig, A. Rehfeldt, and T.J. Jentsch. 1995. Nature (Lond.). 373:527–531). The deactivation at negative voltages of the muscular ClC-1 channel is similar but not identical to ClC-0. Different from the extrinsic voltage dependence suggested for ClC-0, an intrinsic voltage sensor had been proposed to underlie the voltage dependence in ClC-1 (Fahlke, C., R. Rüdel, N. Mitrovic, M. Zhou, and A.L. George. 1995. Neuron. 15:463–472; Fahlke, C., A. Rosenbohm, N. Mitrovic, A.L. George, and R. Rüdel. 1996. Biophys. J. 71:695–706). The gating model for ClC-1 was partially based on the properties of a point-mutation found in recessice myotonia (D136G). Here we investigate the functional effects of mutating the corresponding residue in ClC-0 (D70). Both the corresponding charge neutralization (D70G) and a charge conserving mutation (D70E) led to an inwardly rectifying phenotype resembling that of ClC-1 (D136G). Several other mutations at very different positions in ClC-0 (K165R, H472K, S475T, E482D, T484S, T484Q), however, also led to a similar phenotype. In one of these mutants (T484S) the typical wild-type gating, characterized by a deactivation at negative voltages, can be partially restored by using external perchlorate (ClO₄ ⁻) solutions. We conclude that gating in ClC-0 and ClC-1 is due to similar mechanisms. The negative charge at position 70 in ClC-0 does not specifically confer the voltage sensitivity in ClC-channels, and there is no need to postulate an intrinsic voltage sensor in ClC-channels.