CLC chloride channels and transporters
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;
References (55)
- et al.
Functional and structural analysis of ClC-K chloride channels involved in renal disease
J Biol Chem
(2000) - et al.
Gating competence of constitutively open CLC-0 mutants revealed by the interaction with a small organic Inhibitor
J Gen Physiol
(2003) - et al.
Electrostatics of ion stabilization in a ClC chloride channel homologue from Escherichia coli
J Mol Biol
(2004) - et al.
CBS domains form energy-sensing modules whose binding of adenosine ligands is disrupted by disease mutations
J Clin Invest
(2004) - et al.
Functional evaluation of human ClC-2 chloride channel mutations associated with idiopathic generalized epilepsies
Physiol Genomics
(2004) - et al.
Mutations in CLCN2 encoding a voltage-gated chloride channel are associated with idiopathic generalized epilepsies
Nat Genet
(2003) - et al.
Additional disruption of the ClC-2 Cl− channel does not exacerbate the cystic fibrosis phenotype of CFTR mouse models
J Biol Chem
(2004) - et al.
Regulation of CLC-Ka/barttin by the ubiquitin ligase Nedd4-2 and the serum- and glucocorticoid-dependent kinases
Kidney Int
(2004) - et al.
A common sequence variation of the CLCNKB gene strongly activates ClC-Kb chloride channel activity
Kidney Int
(2004) - et al.
The ClC-5 chloride channel knock-out mouse - an animal model for Dent's disease
Pflugers Arch
(2003)