Hyposmotically activated chloride channels in cultured rabbit non-pigmented ciliary epithelial cells

1. We used whole-cell patch-clamp recording techniques and noise analysis of whole-cell current to investigate the properties of hyposmotic shock (HOS)-activated Cl^- channels in SV40- transformed rabbit non-pigmented ciliary epithelial (NPCE) cells. 2. Under conditions designed to isolate Cl^- currents, exposure of cells to hyposmotic external solution reversibly increased the whole-cell conductance. 3. The whole-cell current activated with a slow time course (> 15 min), exhibited outward rectification and was Cl^- selective. 4. The disulphonic stilbene derivatives 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS, 0.5 mM), 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS, 0.5 mM) and 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS, 0.5 mM) produced a voltage-sensitive block of HOS-activated Cl^- current at depolarized potentials, whereas niflumic acid produced a voltage-independent block of the current. 5. Under Ca²⁺-free conditions, HOS stimulation still reversibly activated the Cl^- current, but the amplitude of current was reduced and the time course of current activation was slower compared with control (P < 0.05). 6. The non-specific kinase inhibitor H-7 (100 μM) upregulated HOS-activated Cl^- current amplitude in all cells tested (P < 0.05). 7. Noise analysis of whole-cell Cl^- current indicated that cell swelling activated a high density of small conductance Cl^- channels (< 1 pS). 8. We conclude that HOS primarily activates a high density of volume-sensitive small conductance Cl^- channels in rabbit NPCE cells, and that Ca²⁺ and phosphorylation are involved in channel regulation.