CLC chloride channels and transporters

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CLC proteins are found in cells from prokaryotes to mammals and perform functions in plasma membranes and intracellular vesicles. Several genetic human diseases and mouse models underscore their broad physiological functions in mammals. These functions range from the control of excitability to transepithelial transport, endocytotic trafficking and acidification of synaptic vesicles. The recent crystallization of bacterial CLC proteins gave surprising insights into CLC Cl-channel permeation and gating and provides an excellent basis for structure–function studies. Surprisingly, the CLC from Escherichia coli functions as a Cl/H+ exchanger, thus demonstrating the thin line separating transporters and channels.

Introduction

The CLC gene family (see glossary) was identified almost 15 years ago [1] by the cloning of ClC-0, a chloride channel enriched in the electric organ of the marine ray Torpedo [2]. Since then, nine mammalian CLC genes have been identified, their products characterized, and prokaryotic CLCs crystallized. Mammalian CLC proteins can be grouped into three branches by homology (see Table 1). Members of the first branch, which includes ClC-1, the ortholog of ClC-0, function as plasma membrane ion channels. Members of the two other branches reside mainly in intracellular membranes. This prevented a biophysical characterization of ClC-6 and ClC-7, but upon overexpression, ClC-3 to -5 yielded plasma membrane currents. However, in the absence of single-channel recordings, and because their extreme rectification precludes determinations of reversal potentials, it is uncertain whether they are Cl-channels or electrogenic Cl/H+ exchangers, as was recently shown for the prokaryotic protein ClC-e1 [3••]. The crystal structures of prokaryotic CLC proteins [4, 5••] provided detailed insights into their structure and function. The broad physiological roles of CLC channels are apparent from mouse models and human genetic diseases, which range from myotonia to renal salt loss, kidney stones, deafness, and osteopetrosis. A recent in-depth review of the physiological functions of CLC proteins is available [6].

Section snippets

The structure and function of CLC proteins

CLC Cl-channels are (homo)dimers in which each of the two subunits has its own pore. This ‘double-barrel’ model was postulated based on the biophysical analysis of reconstituted Torpedo ClC-0 channels [2]. After the cloning of ClC-0 [1], patch-clamp analysis of ClC-0 wild type/mutant [7, 8] and ClC-0/ClC-2 heteromers [9] strongly supported this concept and revealed that a pore is formed entirely within each subunit [9]. The crystal structures of bacterial CLC proteins [4, 5••] confirmed these

ClC-2, a broadly expressed plasma membrane channel

ClC-2 is a plasma membrane channel that is activated by hyperpolarization, cell swelling and mild extracellular acidification. The disruption of this nearly ubiquitous channel in mice caused retinal and testicular degeneration [25]. It was suggested that photoreceptors and germ cells die because the pH in the narrow cleft between these cells and retinal pigment epithelial cells and Sertoli cells, respectively, cannot be regulated properly in the absence of ClC-2. The transepithelial transport

ClC-K/barttin heteromeric chloride channels: transepithelial transport in the kidney and the inner ear

ClC-Ka and ClC-Kb are homologous channels with a high degree of amino acid identity that need the accessory β-subunit barttin for functional expression [12]. Barttin is a  40 kDa protein with two predicted transmembrane domains that is crucial for the transport of ClC-Ka and ClC-Kb to the plasma membrane.

ClC-K/barttin heteromeric channels perform transepithelial transport in the kidney and in the inner ear. ClC-Kb mutations underlie the severe renal salt loss in Bartter syndrome III (see

ClC-3, -4 and -5: endosomal CLCs with diverse functions

ClC-3 to ClC-5 are expressed in endosomes. Upon heterologous expression, they can reach the plasma membrane where they mediate strongly outwardly rectifying, pH-dependent currents [40].

The best known of these proteins is ClC-5. It is expressed in early and recycling endosomes, with particularly increased levels in subapical compartments of the renal proximal tubule (PT). The role of ClC-5 in endocytosis is evident from the proteinuria in Dent's disease (an inherited kidney stone disorder caused

Disruption of lysosomal ClC-7 leads to osteopetrosis and lysosomal storage disease

In contrast to the other vesicular CLC proteins, which are predominantly expressed on endosomes, ClC-7 is mainly found on lysosomes and late endosomes [52, 53•]. ClC-7 is ubiquitously expressed [52, 53•]. In osteoclasts, the cells that degrade bone, ClC-7 is also present in the ‘ruffled border’. It can be inserted into this specialized plasma membrane domain together with a V-type ATPase by an exocytotic insertion from late endosomal–lysosomal membranes.

The disruption of ClC-7 resulted in

Conclusions

The combination of broad physiological functions and roles in disease, the availability of crystal structures providing an excellent basis for structure–function studies, and the unexpected finding that prokaryotic CLCs function as coupled transporters guarantee that the CLC field will continue to generate excitement. Future work will improve our understanding of permeation and gating, clarify the structural basis for transporter versus channel activity, and address the roles of CBS domains. It

References and recommended reading

Papers of particular interest, published within the annual period of review, have been highlighted as:

  • • of special interest

  • •• of outstanding interest

Glossary

Bartter syndrome
Hereditary kidney diseases that are associated by a massive renal salt loss. The syndrome is genetically heterogeneous, with Bartter syndrome III caused by mutations in CLCNKB, the gene encoding the ClC-Kb α-subunit, and Bartter syndrome IV caused by mutations in BSND, the gene encoding the barttin β-subunit. Bartter IV is additionally associated with congenital deafness.
CBS domains
Protein domains that are named after cystathionine β-synthase, a protein in which they are found;

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