Quantifying the mean negative shift in V _h following injection of a 1:1 mixture of G375R mutant and WT cRNA when compared to injection of only WT cRNA. Data are for essentially 0 Ca²⁺ at the intracellular side of the membrane for Figs. 3, 4, 5, and 6. (A) Currents recorded from inside-out macro patches of membrane excised from oocytes. For WT channels following injection of only WT cRNA, the holding potential was −80 mV and voltage pulses were from −80 to 240 mV with 20 mV steps followed by a step back to −80 mV to elicit tail currents for G/G_max measurements. For channels expressed following injection of a 1:1 mixture of G375R mutant and WT cRNA, the holding potential was −160 mV and voltage pulses were from −120 to 240 mV with 20 mV steps followed by a step back to −120 mV to elicit tail current for measurement. The currents are the mean response from the many hundreds to thousands of BK channels in each excised macropatch of membrane. (B) G-V plots following the injection of WT cRNA alone (black circles, n = 6; mean ± SEM), or 1:1 injection of mutant and WT cRNA (red diamonds, n = 12; mean ± SEM). The black line through the WT data is a single Boltzmann function with V_h = 181.3 mV and slope factor b = 24.04 mV/e-fold change in Po. The blue line through the 1:1 data (red diamonds) is a single Boltzmann function with V_h = 59.04 mV and b = 38.06 mV/e-fold change. The red line through the 1:1 data is the sum of five Boltzmann functions with (1) V_h = −75.7 mV, b = 21.6 mV/e-fold change, fractional area = 0.0547; (2) 1.5, 21.0, 0.236; (3) 54.6, 13.8, 0.316; (4) 96.6, 14.4, 0.244; (5) 155, 14.0, 0.139. These parameters for the five summed Boltzmann functions that describe the 1:1 data are generally consistent with the V_h values and percentages for the apparent clusters of assembled channels (Fig. 5, A and B) and theoretical percentages in Fig. 1.