Figure 8.

Whole-cell patch-clamp analysis of HEK293 cells expressing TMEM16B. A, B, Representative voltage-clamp recordings from a HEK293 cell transfected with YFP (A) and YFP and TMEM16B (B) stimulated with a ramp protocol shown in the inset in A. Membrane potentials were clamped at a holding potential of −40 mV and the cells were depolarized to 50 mV in 10 mV steps in 50 ms followed by −10 mV steps to −130 mV in 50 ms. Intracellular Ca²⁺ concentration was 100 nm. C, Current–voltage relationships from experiments shown in A and B. The currents in the I–V plots are expressed as current densities which were calculated as described previously (Wesolowski et al., 2007). Each value represents the mean current densities (± SEM) measured from cells at each voltage pulse. D–G, Representative voltage-clamp recordings and current–voltage relationships from HEK293 cells transfected with YFP (D, E) and YFP and TMEM16B (F, G) obtained by the protocol shown in the inset in D. The membrane currents were continuously recorded at a holding potential of −40 mV for 125 s. During this time the cells were repeatedly stimulated every 2.5 s by −20 mV voltage steps (each 100 ms) to −140 mV followed by depolarization steps to 60 mm. Treatment with 1 μm ionomycin is indicated by black bars. The mean current densities (± SEM) of cells with and without ionomycin application (control) were plotted against the test potentials of the electrical stimulation. Insets in F show magnifications of current deflections obtained with HEK293 cells expressing TMEM16B before and after ionomycin application. H, I, Current–voltage relationships from HEK293 cells transfected with YFP and TMEM16B obtained with Ringer or low-Cl⁻-Ringer solution by the protocol shown in A. Current–voltage relationship in I was measured after ionomycin application. Replacing Ringer bath solution with symmetric Cl⁻ reduced current amplitudes and caused statistically significant shifts of the reversal potentials to positive values (p < 0.001) (J).