G375R homotetrameric mutant channels, assembled channels, and WT BK channels are expressed in HEK293 cells. (A) Whole-cell currents were recorded from HEK293 cells following transfection with the indicated types of cDNA. The whole-cell currents were generated by holding at −60 mV, with a prestep to −100 mV, followed by steps from either −200 or −100 to +200 mV in 10 mV increments followed by a post-step to −120 mV. The dashed line indicates the level of 0 current. Evoked currents were not observed in HEK293 cells that were not transfected. (B) Plots of normalized conductance versus the voltage of the activating steps following transfections with G375R mutant cDNA (blue squares); a 1:1 mixture of mutant and WT cDNA (red diamonds) to express assembled channels; or WT cDNA (black circles). Unlike single-channel recordings (Fig. 4), whole-cell recordings reflect the average response from many hundreds to thousands of channels. There were large negative shifts in V_h in the G-V curves determined from G375R homotetrameric mutant channels (blue) and from assembled channels (red) compared with WT channels (black). The mean V_h was 130.3 ± 4.4 mV (n = 4) for WT BK currents, 37.5 ± 2.7 mV (n = 5) for BK currents following the 1:1 injection, and −142.5 ± 3.6 mV (n = 3) for the G375R homotetrameric mutant channels. The 1:1 transfection also decreased the voltage sensitivity of activation compared with WT, with a slope of 52.0 ± 2.0 mV (n = 5) per e-fold change for current after a 1:1 transfection, compared with 32.3 ± 2.5 mV (n = 4) for WT currents. The G375R homotetrameric mutant channels had a slope of 45.0 ± 4.0 mV. Mean ± SEM.