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PERSPECTIVES |
Department of Basic Medical Sciences, Pharmacology and Clinical Pharmacology, St George's Hospital Medical School, London, UK
Email: i.greenwood{at}sghms.ac.uk
Chloride currents activated as a consequence of mechanical stress such as cell swelling or membrane stretch have been recorded in a wide range of cell types that include cardiomyocytes, endothelial cells, epithelial cells and smooth muscle cells. Whilst considerable information on the biophysical and pharmacological properties of the underlying native channels has been accrued, the molecular nature remains elusive due primarily to the lack of a specific, high affinity ligand and the presence of swelling-activated Cl– currents in most cells studied. Various candidates have been proposed and in turn subsequently dismissed (see Jentsch et al. 2002; Nilius & Droogmans, 2003). The most recent contender has been a member of the CLCN gene family of voltage-gated Cl– channels, CLC-3. The case for CLC-3 as the molecular correlate of swelling-activated Cl– currents (which for the benefit of this article will be abbreviated to Iswell although various nomenclature exist) was based upon a number of observations. Heterologous expression of guinea-pig CLC-3 in NIH/3T3 cells generated an outwardly rectifying Cl– current with many biophysical and pharmacological properties of native Iswell. In addition commercially available antibodies raised against the CLC-3 protein inhibited native Iswell and antisense oligonucleotides significantly inhibited Iswell in epithelial and HeLa cells. Whilst the evidence for CLC-3 as a contender for the channel underlying Iswell seemed quite persuasive, the putative involvement of CLC-3 has been disputed on a number of lines. Firstly, a number of laboratories have failed to show swelling-activated Cl– currents due to the over-expression of CLC-3 (Weylandt et al. 2001; Jentsch et al. 2002), although Cl– currents generated by the heterologous expression of the closely related genes CLC-4 and CLC-5 have been reported by these groups. Secondly, CLC-3-induced currents have an anion permeability that differs from that of channels encoded by the closely related genes CLC-4 and CLC-5 (Weylandt et al. 2001; Jentsch et al. 2002). In addition the commercially available anti-CLC-3 antibody was shown to cross-react with a number of proteins. Furthermore, in those studies that reported Cl– currents due to CLC-3 over-expression, large Cl– currents were recorded in isotonic conditions in the absence of any cell swelling. This was in distinct comparison to native Iswell where very little Cl– activity is observed in non-bloated cells. Finally, CLC-3 proteins were observed to localize primarily in the cytoplasm with limited plasmalemmal staining (Weylandt et al. 2001). These observations led to the proposal that CLC-3 encoded a protein that was normally stored within cytoplasmic vesicles that, when over-expressed, augmented the amplitude of Iswell endogenous to the cell type under investigation. The final nail in the CLC-3 coffin was the generation of a global CLC-3 gene disrupted (Clcn3–/–) mouse that exhibited native Iswell that were indistinguishable from Iswell in wild-type (Clcn-3+/+) animals (Stobrawa et al. 2001).
The paper by Yamamoto-Mizuma et al. (2004) in this issue of TheJournal of Physiology resurrects the case for CLC-3 contributing to the channels underlying native Iswell (referred to as volume-sensitive osmolyte and anion channels, VSOACs). The authors show that in terms of current density, rectification, anion selectivity and sensitivity to various blockers there was no discernable difference in Iswell recorded in atrial cells from Clcn3+/+ and Clcn3–/– animals. However, in comparison to currents from wild-type animals, Iswell in cells from Clcn3–/– mice were not inhibited by phorbol esters, increased intracellular Mg2+, reduced intracellular ATP or application of two new CLC-3 antibodies (raised against amino acids 1–14 and 670–687). Furthermore, proteomic analysis revealed that 35 distinct membrane proteins underwent significant changes in expression levels in the Clcn3–/– animals.
The observations by Yamamoto-Mizuma et al. (2004) highlight that considerable alterations in untargeted protein expression can occur with global gene knockout models and therefore conclusions drawn from these transgenic animals, whilst obviously powerful, must be treated with caution. However, the study suggests that dismissal of CLC-3 as a candidate for Iswell may have been premature but the original hypothesis needed refinement. The data presented do not answer the elusive question of what constitutes the Iswell chloride channel but implicate CLC-3-encoded proteins as a component of the channel complex, and this suggests intuitively that the channel underlying Iswell is a heteromultimer. Moreover data presented by Yamamoto-Mizuma et al. (2004) support the existence of distinct subpopulations of Iswell chloride channels that can be discriminated by their sensitivity to PKC modulators and anti-CLC-3 antibodies. Duan et al. (1999) formulated a hypothesis that Iswell was activated by cell swelling as a consequence of protein dephosphorylation at PKC consensus sites based upon mutational analysis of heterologously expressed CLC-3 and the inhibitory effect of PKC activators on native Iswell. This cannot be a ubiquitous gating mechanism as there are a number of cell types where PKC activators increase the amplitude of Iswell (e.g. rabbit portal vein; Ellershaw et al. 2002). Interestingly a lack of inhibition by PKC in the Clcn3–/– animals was allied to a loss of sensitivity to anti-CLC-3 antibodies. Therefore it appears that the presence of CLC-3 affects the regulation of the swelling-activated current rather than the intrinsic pore properties (anion selectivity, pharmacology). The study by Yamamoto-Mizuma et al. (2004) has not revealed the identity of swelling-activated Cl– channels but has given us a glimpse that the elusive channel may be more complicated and variable than considered initially. It remains a future challenge to identify the proteins involved and to put the jigsaw together.
References
Duan D, Cowley S, Horowitz B & Hume JR (1999). J General Physiol 113, 57–70.
Ellershaw DC, Greenwood IA & Large WA (2002). J Physiol 542, 537–547.
Jentsch TJ, Stein V, Weinreich F & Zdebik AA (2002). Physiol Rev 82, 503–568.
Nilius B & Droogmans G (2003). Acta Physiol Scand 177, 119–147.[CrossRef][Medline]
Stobrawa SM, Breiderhof T, Takamori S, Engel D, Schweizer M, Zdebik AA, Bösl MR, Ruether K, Jain H, Draguhn A, Jain R & Jentsch TJ (2001). Neuron 29, 185–196.[CrossRef][Medline]
Weylandt KH, Valverde MA, Nobles M, Raguz S, Amey JS, Diaz M, Nastrucci C, Higgens CF & Sardini A (2001). J Biol Chem 276, 17461–17467.
Yamamoto-Mizuma S, Wang GX, Liu LL, Schegg K, Hatton WJ, Duan D, Horowitz B, Lamb FS & Hume JR (2004). J Physiol 557, 443–460.
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