A role for the volume regulated anion channel in volume regulation in the murine CNS cell line,CAD

Aim: The role of the volume regulated anion channel (VRAC) in a model CNS neuronal cell line, CAD, was investigated. Methods: Changes in cell volume following hypotonic challenges were measured using a video-imaging technique. The effect of the Cl⁻ channel antagonists tamoxifen (10 μm ) and 4,4′-diisothiocyanatostilbene-2,2′-disulphonic acid (DIDS; 100 μm ) on regulatory volume decrease (RVD) were measured. The whole-cell voltage-clamp technique was used to characterize ICl_swell, the current underlying the VRAC. Results: Using the video-imaging technique, CAD cells were found to swell and subsequently exhibit RVD when subjected to a sustained hypotonic challenge from 300 mOsmol kg⁻¹ H₂O to 210 mOsmol kg⁻¹ H₂O. In the presence of tamoxifen (10 μm ) or DIDS (100 μm ) RVD was abolished, suggesting a role for the VRAC. A hypotonic solution (230 mOsmol kg⁻¹ H₂O) evoked ICl_swell, an outwardly rectifying current displaying time-independent activation, which reversed upon return to isotonic conditions. The reversal potential (E_rev) for ICl_swell was −14.7 ± 1.4 mV, similar to the theoretical E_rev for a selective Cl⁻ conductance. ICl_swell was inhibited in the presence of DIDS (100 μm ) and tamoxifen (10 μm ), the DIDS inhibition being voltage dependent. Conclusions: Osmotic swelling elicits an outwardly rectifying Cl⁻ conductance in CAD cells. The ICl_swell observed in these cells is similar to that observed in other cells, and is likely to provide a pathway for the loss of Cl⁻ which leads to water loss and RVD. As ischaemia, brain trauma, hypoxia and other brain pathologies can cause cell swelling, CAD cells represent a model cell line for the study of neuronal cell volume regulation.