Rescue of the volume-regulated Cl⁻ current in S2 cells by expression of dBest1-F81C. Endogenous dBest1 in S2 cells was knocked down by RNAi that targeted the 5′ UTR of dBest1 for 4 d. Cells were then transfected with dBest1-F81C and EGFP expression constructs and patch clamped in whole cell configuration with 10-s interval voltage ramps from −100 mV to +100 mV from a holding potential of 0 mV. Currents recorded from native S2 cells, dBest1 RNAi-treated S2 cells, and F81C overexpressed in dBest1 RNAi-treated S2 cells are labeled as native, RNAi, and F81C, respectively. (A) Current–voltage relationship of dBest1 currents in native and F81C-rescued cells recorded with Δ0 mosmol kg⁻¹ isosmotic solutions. (B) Current–voltage relationship of dBest1 currents in native, F81C-rescued, and RNAi-only S2 cells stimulated with Δ40 mosmol kg⁻¹ hyposmotic solutions. (C) Time course of activation of VRAC in S2 cells rescued with F81C. The currents shown were measured at −100 mV (open symbols) and +100 mV (solid symbols) under isosmotic (I300/E300, triangles; n = 6) and hyposmotic (I340/E300, circles; n = 7) conditions. (D) Mean current amplitudes at 100 mV at the onset of whole cell recording (initial current, open bars) and after the currents had reached a peak (final currents, filled bars) and the corresponding cell volume alterations (hatched bars) with hyposmotic (Δ40 mosmol kg⁻¹) and isosmotic solutions. Changes in cell volume are expressed as percent change in cell volume from the initiation of whole cell recording to ∼3–5 min after patch break (isosmotic; n = 6) or when the currents had approached a steady value (hyposmotic; n = 7). Data are represented in mean ± SEM. *, significantly different at P < 0.05 and ** at P < 0.01 level.