Criteria for the Molecular Identification of the Volume-Sensitive Outwardly Rectifying Cl− Channel

Anion channels are activated by volume expansion in most animal cell types and are known to be implicated not only in regulatory volume decrease but also in many other cell activities that are associated with changes in cell volume and shape, including cell proliferation and cell death (Okada, 1998). Among a variety of swellingactivated Cl 2 channels (I Cl.swell ), the most important and frequently observed channel is a volume-sensitive, outwardly rectifying, ATP-dependent Cl 2 channel (Okada, 1997; termed VSOR-ClC here). The functional properties of VSOR-ClC have so far been intensively and systematically studied in rat C6 glioma cells (Strange et al., 1996), human epithelial Intestine 407 cells (Okada, 1997), and human and bovine endothelial cells (Nilius et al., 1997), and proved to share the same properties. The principal phenotypic properties are attributed to the pore and regulatory characteristics, as summarized in Table I. The regulatory properties of the channel may be modified by association with putative regulatory subunits. Thus, the “fingerprint” characteristics of the channel, which distinguish it from other Cl 2 channels, should be related, at least, to the pore properties, such as its intermediate single-channel conductance, outward rectification, low-field anion selectivity, a high open probability ( . 0.95), voltage-dependent inactivation gating at large positive potentials, and open-channel block by extracellular ATP. Although much work has been performed to identify the VSOR-ClC protein, the molecular identity of the channel has not as yet been determined. Purification of the channel protein has been hindered by the lack of a highly specific channel ligand (antagonist or agonist). Expression cloning of the VSOR-ClC protein has been hampered by the endogenous expression and housekeeping activity of the channel in almost all cell types (Nilius et al., 1994; Okada, 1997), including Xenopus oocytes (Ackerman et al., 1994), and by possible changes in volume sensitivity of the channel and/or the osmosensitivity of the cell produced by overexpression of exogenous proteins (Okada, 1997). From gene expression studies, three candidates for the VSOR-ClC protein have been proposed: the MDR1 gene product, P-glycoprotein (PGP; Valverde et al., 1992; Gill et al., 1992), a ubiquitous (principally cytoplasmic) protein, pICln (Paulmichl et al., 1992), and a member of the cloned ClC family, ClC-3 (Duan et al., 1997). However, the PGP hypothesis is no longer viable, as reviewed by Okada (1997). The most crucial evidence against the PGP hypothesis was the observation that abolition of the endogenous PGP expression by antisense oligonucleotides failed to affect the VSOR-ClC current in Intestine 407 cells (Tominaga et al., 1995). The misleading conclusion in the original studies (Valverde et al., 1992; Gill et al., 1992) may have been caused by exaggerated experimental conditions, because overexpression of PGP was found to augment volume sensitivity of endogenous VSOR-ClC currents (Miwa et al., 1997). Now, the pICln hypothesis has also been discarded, as summarized recently in two letters for Perspectives in General Physiology (Strange, 1998; Clapham, 1998). Several lines of crucial evidence against the pICln hypothesis were provided by Voets et al. (1996, 1998), Buyse et al. (1997), and Emma et al. (1998). It should be stressed that overexpression of pICln in Xenopus oocytes resulted in activation of Cl 2 currents with pore properties (rectification, anion selectivity, and blocking) distinct from the phenotypic properties of VSOR-ClC (Voets et al., 1996). The ClC-3 hypothesis is at present viable, though not completely tested. As summarized in Table I, the pore properties, except the open probability, of guinea pig cardiac ClC-3-associated Cl 2 currents (Duan et al., 1997) are largely similar to those of VSOR-ClC. Interestingly, the N579K mutant of guinea pig ClC-3 was found to bring about changes in anion selectivity from I 2 . Cl 2