The aberrant negative shift in V _h for BK currents expressed following a 1:1 injection of G375R mutant and WT cRNA is also observed with 300 μM intracellular Ca ²⁺ . (A) Currents recorded from inside-out macro patches of the membrane with 300 μM Ca²⁺ at the inner membrane surface. For WT channels, voltage pulses were from −160 to 180 mV with 20 mV steps from a holding potential of −160 mV. Conductance was measured from tail currents after stepping to −160 mV. For channels expressed following injection of a 1:1 mixture of G375R mutant and WT cRNA, voltage pulses were from −200 to 180 mV with 20 mV steps from a holding potential of −200 mV. Conductance was measured from tail currents after stepping to −200 mV. The dashed line indicates the level of 0 current. In the presence of 300 μM intracellular Ca²⁺, more than half of the channels expressed following the 1:1 injection of G375R mutant and WT cRNA remain open at −200 mV. The macrocurrents are the average response from many hundreds of BK channels in each macro patch. (B) G-V plots with 300 μM Ca²⁺ following injection of WT cRNA alone (black circles) or a 1:1 injection of mutant and WT cRNA (red diamonds). The mean V_h for WT currents was −7.2 ± 3.3 mV, n = 8, shifting to −205 ± 9.6 mV for BK currents expressed from a 1:1 mixture of mutant and WT cRNA, producing a left shift of −198 mV with 300 μM Ca²⁺ at the inner membrane surface, n = 6. This can be compared with a left shift of −120 mV with 0 Ca²⁺ (Fig. 3). Hence, the aberrant left shift in V_h induced by a 1:1 injection of mutant and WT cRNA occurs in the presence and absence of intracellular Ca²⁺.