Michael Pusch

Chloride/proton antiporter activity of mammalian CLC proteins ClC-4 and ClC-5.

ClC-4 and ClC-5 are members of the CLC gene family, with ClC-5 mutated in Dents disease, a nephropathy associated with low-molecular-mass proteinuria and eventual renal failure. ClC-5 has been proposed to be an electrically shunting Cl- channel in early endosomes, facilitating intraluminal acidification. Motivated by the discovery that certain bacterial CLC proteins are secondary active Cl-/H+ antiporters, we hypothesized that mammalian CLC proteins might not be classical Cl- ion channels but might exhibit Cl--coupled proton transport activity. Here we report that ClC-4 and ClC-5 carry a substantial amount of protons across the plasma membrane when activated by positive voltages, as revealed by measurements of pH close to the cell surface. Both proteins are able to extrude protons against their electrochemical gradient, demonstrating secondary active transport. H+, but not Cl-, transport was abolished when a pore glutamate was mutated to alanine (E211A). ClC-0, ClC-2 and ClC-Ka proteins showed no significant proton transport. The muscle channel ClC-1 exhibited a small H+ transport that might be physiologically relevant. For ClC-5, we estimated that Cl- and H+ transport contribute about equally to the total charge movement, raising the possibility that the coupled Cl-/H+ transport of ClC-4 and ClC-5 is of significant magnitude in vivo.

Properties of voltage-gated chloride channels of the ClC gene family.

We review the properties of ClC chloride channels, members of an expanding gene family originally discovered by the cloning of the ClC‐0 chloride channel from Torpedo electric organ. There are at least nine different ClC genes in mammals, several of which seem to be expressed ubiquitously, while others are expressed in a highly specific manner (e.g. the muscle‐specific ClC‐1 channel and the kidney‐specific ClC‐K channels). The newly cloned rat ClC‐4 is strongly expressed in liver and brain, but also in heart, muscle, kidney and spleen. ClC chloride channels are structurally unrelated to other channel proteins and have twelve putative transmembrane domains. They function as multimers with probably four subunits. Functional characterization is most advanced with ClC‐0, ClC‐1 (mutations which cause myotonia) and ClC‐2, a swelling‐activated chloride channel. Many of the new ClC family members cannot yet be expressed functionally.

Multimeric structure of ClC-1 chloride channel revealed by mutations in dominant myotonia congenita (Thomsen).

Voltage‐gated ClC chloride channels play important roles in cell volume regulation, control of muscle excitability, and probably transepithelial transport. ClC channels can be functionally expressed without other subunits, but it is unknown whether they function as monomers. We now exploit the properties of human mutations in the muscle chloride channel, ClC‐1, to explore its multimeric structure. This is based on analysis of the dominant negative effects of ClC‐1 mutations causing myotonia congenita (MC, Thomsens disease), including a newly identified mutation (P480L) in Thomsens own family. In a co‐expression assay, Thomsens mutation dramatically inhibits normal ClC‐1 function. A mutation found in Canadian MC families (G230E) has a less pronounced dominant negative effect, which can be explained by functional WT/G230E heterooligomeric channels with altered kinetics and selectivity. Analysis of both mutants shows independently that ClC‐1 functions as a homooligomer with most likely four subunits.

Molecular determinants of KCNQ (Kv7) K+ channel sensitivity to the anticonvulsant retigabine.

Epilepsy is caused by an electrical hyperexcitability in the CNS. Because K+ channels are critical for establishing and stabilizing the resting potential of neurons, a loss of K+ channels could support neuronal hyperexcitability. Indeed, benign familial neonatal convulsions, an autosomal dominant epilepsy of infancy, is caused by mutations in KCNQ2 or KCNQ3 K+ channel genes. Because these channels contribute to the native muscarinic-sensitive K+ current (M current) that regulates excitability of numerous types of neurons, KCNQ (Kv7) channel activators would be effective in epilepsy treatment. A compound exhibiting anticonvulsant activity in animal seizure models is retigabine. It specifically acts on the neuronally expressed KCNQ2-KCNQ5 (Kv7.2-Kv7.5) channels, whereas KCNQ1 (Kv7.1) is not affected. Using the differential sensitivity of KCNQ3 and KCNQ1 to retigabine, we constructed chimeras to identify minimal segments required for sensitivity to the drug. We identified a single tryptophan residue within the S5 segment of KCNQ3 and also KCNQ2, KCNQ4, and KCNQ5 as crucial for the effect of retigabine. Furthermore, heteromeric KCNQ channels comprising KCNQ2 and KCNQ1 transmembrane domains (attributable to transfer of assembly properties from KCNQ3 to KCNQ1) are retigabine insensitive. Transfer of the tryptophan into the KCNQ1 scaffold resulted in retigabine-sensitive heteromers, suggesting that the tryptophan is necessary in all KCNQ subunits forming a functional tetramer to confer drug sensitivity.

Mutations in dominant human myotonia congenita drastically alter the voltage dependence of the CIC-1 chloride channel

Autosomal dominant myotonia congenita (Thomsens disease) is caused by mutations in the muscle chloride channel CIC-1. Several point mutations found in affected families (I29OM, R317Q, P480L, and Q552R) dramatically shift gating to positive voltages in mutant/WT heterooligomeric channels, and when measurable, even more so in mutant homooligomers. These channels can no longer contribute to the repolarization of action potentials, fully explaining why they cause dominant myotonia. Most replacements of the isoleucine at position 290 shift gating toward positive voltages. Mutant/WT heterooligomers can be partially activated by repetitive depolarizations, suggesting a role in shortening myotonic runs. Remarkably, a human mutation affecting an adjacent residue (E291K) is fully recessive. Large shifts in the voltage dependence of gating may be common to many mutations in dominant myotonia congenita.

Conservation of Chloride Channel Structure Revealed by an Inhibitor Binding Site in ClC-1

Crystal structures of bacterial CLC proteins were solved recently, but it is unclear to which level of detail they can be extrapolated to mammalian chloride channels. Exploiting the difference in inhibition by 9-anthracene carboxylic acid (9-AC) between ClC-0, -1, and -2, we identified a serine between helices O and P as crucial for 9-AC binding. Mutagenesis based on the crystal structure identified further residues affecting inhibitor binding. They surround a partially hydrophobic pocket close to the chloride binding site that is accessible from the cytoplasm, consistent with the observed intracellular block by 9-AC. Mutations in presumably Cl--coordinating residues yield additional insights into the structure and function of ClC-1. Our work shows that the structure of bacterial CLCs can be extrapolated with fidelity to mammalian Cl- channels.

Functional and structural conservation of CBS domains from CLC chloride channels

All eukaryotic CLC Cl− channel subunits possess a long cytoplasmic carboxy‐terminus that contains two so‐called CBS (cystathionine β‐synthase) domains. These domains are found in various unrelated proteins from all phylae. The crystal structure of the CBS domains of inosine monophosphate dehydrogenase (IMPDH) is known, but it is not known whether this structure is conserved in CLC channels. Working primarily with ClC‐1, we used deletion scanning mutagenesis, coimmunoprecipitation and electrophysiology to demonstrate that its CBS domains interact. The replacement of CBS domains of ClC‐1 with the corresponding CBS domains from other CLC channels and even human IMPDH yielded functional channels, indicating a high degree of structural conservation. Based on a homology model of the pair of CBS domains of CLC channels, we identified some residues that, when mutated, affected the common gate which acts on both pores of the dimeric channel. Thus, we propose that the structure of CBS domains from CLC channels is highly conserved and that they play a functional role in the common gate.

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.

Chloride dependence of hyperpolarization‐activated chloride channel gates

1 ClC proteins are a class of voltage‐dependent Cl− channels with several members mutated in human diseases. The prototype ClC‐0 Torpedo channel is a dimeric protein; each subunit forms a pore that can gate independently from the other one. A common slower gating mechanism acts on both pores simultaneously; slow gating activates ClC‐0 at hyperpolarized voltages. The ClC‐2 Cl− channel is also activated by hyperpolarization, as are some ClC‐1 mutants (e.g. D136G) and wild‐type (WT) ClC‐1 at certain pH values. 2 We studied the dependence on internal Cl− ([Cl−]i) of the hyperpolarization‐activated gates of several ClC channels (WT ClC‐0, ClC‐0 mutant P522G, ClC‐1 mutant D136G and an N‐terminal deletion mutant of ClC‐2), by patch clamping channels expressed in Xenopus oocytes. 3 With all these channels, reducing [Cl−]i shifted activation to more negative voltages and reduced the maximal activation at most negative voltages. 4 We also investigated the external halide dependence of WT ClC‐2 using two‐electrode voltage‐clamp recording. Reducing external Cl− ([Cl−]o) activated ClC‐2 currents. Replacing [Cl−]o by the less permeant Br− reduced channel activity and accelerated deactivation. 5 Gating of the ClC‐2 mutant K566Q in normal [Cl−]o resembled that of WT ClC‐2 in low [Cl−]o, i.e. channels had a considerable open probability (Po) at resting membrane potential. Substituting external Cl− by Br− or I− led to a decrease in Po. 6 The [Cl−]i dependence of the hyperpolarization‐activated gates of various ClC channels suggests a similar gating mechanism, and raises the possibility that the gating charge for the hyperpolarization‐activated gate is provided by Cl−. 7 The external halide dependence of hyperpolarization‐activated gating of ClC‐2 suggests that it is mediated or modulated by anions as in other ClC channels. In contrast to the depolarization‐activated fast gates of ClC‐0 and ClC‐1, the absence of Cl− favours channel opening. Lysine 556 may be important for the relevant binding site.

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-4E281A and ClC-5E268A 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. \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{NO}_{3}^{-}\) \end{document} 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.