Effects of Ca ²⁺ -site mutations on BK channel modulation by γ subunits. (A) Representative traces of recorded BK channel currents in response to depolarization of the membrane potential from −80 mV in 20-mV steps for selected expression constructs in the virtual absence of [Ca²⁺]_i. (B–D) Voltage dependence of BK channel activation for channels formed by BKα mutant (D362A/D367A, 5D5N, or D362A/D367A/5D5N) with and without γ1 (B), γ2 (C), or γ3 (D) subunit in the virtual absence of [Ca²⁺]_i. For comparison, the voltage dependence of WT BK channel activation in the absence and presence of the γ subunit was included in B–D. (E) Summary of the BKα mutation-induced reduction (ΔΔV_1/2) in the V_1/2-shifting capabilities (ΔV_1/2) of the γ1, γ2, and γ3 subunits (n = 4–10). Triple mutant: BKα (D362A/D367A/5D5N). The data obtained with the channel complex of BKα (triple mutant) and γ1 were fitted with a double Boltzmann function. (F) Immunoprecipitation of the BKα subunit (WT and 5D5N mutant) with the γ1 subunit. Error bars represent ± SEM. Source data are available for this figure: SourceData F6.