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 ).