The mechanism and state dependence of Cu²⁺ action. (A) Mean log(P_o)-V relations for mSlo1 for 0 and 5 μM [Ca²⁺]_i in 0 Cu²⁺(○) and 100 μM Cu²⁺(▪), respectively. Dotted lines are exponential fits to the limiting slope of log(P_o) with partial charge z_L = 0.3 e. (B) Log(P_o)-V relations from A are superimposed by shifting the 0 Cu²⁺ curves along the voltage axis by +45 mV. (C) I_K evoked by 30-ms test pulses to +120 mV before (I_P1) and after (I_P2) application of 100 μM Cu²⁺ using the illustrated protocol (5 μM [Ca²⁺]_i), without leak subtraction. I_P1 was evoked from a holding potential of −80 mV in 0 Cu²⁺ following perfusion with standard external solution for 5 s. I_P2 was recorded in 100 μM Cu²⁺ following perfusion with 100 μM Cu²⁺ for 500 ms at different prepulse voltages (V_PRE). Following the second pulse the patch was washed at −80 mV for 5 s with 5 mM EGTA and then for 5 s with standard external solution before repeating the protocol. Maximal inhibition of I_P2 was observed with V_PRE = −80 to +40 mV (green traces) and minimal inhibition with V_PRE = +180 to +220 mV (red traces). (D) The fraction of channels not inhibited f_NI = (I_P2[V_PRE] − I_P2[−80])/(I_P1 − I_P2[−80]) from C (▪, f_NI[100 Cu²⁺]) is plotted against V_PRE and compared with the mean steady-state P_o-V relation (○) in 0 Cu²⁺ and 5 μM [Ca²⁺]_i estimated as G_K/G_Kmax. A control experiment (□, f_NI[0 Cu²⁺]) obtained from a different patch using the pulse protocol in C shows that f_NI is voltage independent when the 100 μM Cu²⁺ solution is replaced with 0 Cu²⁺.