Using the whole-cell configuration of the patch-clamp technique and video-imaging analysis, we have recently reported activation of the volume-regulated anion channel (VRAC) in response to hypotonic stress (Harvey et al. 2002, 2003). In the present study, we have further characterised the VRAC and investigated its molecular identity.

Whole-cell patch-clamp studies were performed as previously reported (Harvey et al. 2003). Data reported below were obtained by 100 ms step depolarisations from -80 to +100 mV from a holding potential of -60 mV, and are expressed as mean ± S.E.M. current densities (pA pF^-1). Significant differences were determined using Student’s paired t test or ANOVA followed by the Tukey HSD test.

Cells were bathed in isotonic bath solution containing (mM): NaCl 110, CaCl₂ 1, MgCl₂ 1, Hepes 10, adjusted with NaOH (pH 7.3) and mannitol (300 mosmol (kg H₂O)^-1). They were dialysed with a pipette solution containing (mM): NaCl 50, EGTA 1, MgCl₂ 1, Hepes 10, adjusted with NaOH (pH 7.3) and mannitol (290 mosmol (kg H₂O)^-1). Under these conditions CAD cells displayed a maximum outward current of 8.4 ± 2.4 pA pF^-1 and maximum inward current of -0.30 ± 0.44 pA pF^-1 (n = 16). After a 5 min exposure to an iso-ionic hypotonic solution (230 mosmol (kg H₂O)^-1), the outward current increased to 48.2 ± 6.5 pA pF^-1 and the inward current increased to -15.0 ± 3.0 pA pF^-1 (n = 16, P < 0.001). The reversal potential (E_rev), -14.7 ± 1.4 mV (n = 16), was similar to the theoretical E_rev for a Cl^–-selective conductance under these conditions. Some of the data above have previously been reported (Harvey et al. 2003). Bath perfusion with the Cl^– channel antagonist tamoxifen (10 µM) reduced the outward current (56.4 ± 15.6 pA pF^-1) to 22.1 ± 4.8 pA pF^-1 (n = 4, P < 0.05), but had no significant effects on the inward current. This inhibition was reversed following washout with hypotonic solution (53.8 ± 26.3 pA pF^-1, n = 4). The molecular identity of the VRAC is controversial, with two members of the voltage-dependent Cl^– family, ClC-2 and ClC-3, proposed as the most likely candidates. The expression of members of the ClC family was examined in CAD cells and liver tissue, isolated from humanely killed rats, using RT-PCR. Using primers specific for ClC-1, -2, -3, -4 and -5 (ClC-2, ClC-3; Enz et al. 1999), transcripts for ClC-2, -3, -4 and -5 were identified in liver; but only ClC-3, -4 and -5 were identified in CAD cells.

In conclusion, CAD cells possess swelling-activated currents that display properties consistent with those of the VRAC reported in other cell types. Furthermore, the biophysical properties of the VRAC, and RT-PCR analysis suggest that ClC-2 does not represent the molecular candidate for the VRAC in CAD cells.