Reversible inhibition by cytoplasmic Cu²⁺ ions. (A) Example leak-subtracted I-V relationships for cys-less (left), I344C (center), and Q98C/I344C (right) after channel activation with 20 nM PKA and 1 mM ATP. In each panel, currents are shown before (control) and after the addition of 10 µM Cu²⁺ to the intracellular solution. In all channel constructs studied, these inhibitory effects of Cu²⁺ were readily and rapidly reversed by washing Cu²⁺ from the bath (for example, see right panel for complete reversal of the strong blocking effect on Q98C/I344C). (B) Mean fractional current remaining after the addition of different concentrations of Cu²⁺ for cys-less (•), I344C (○), and Q98C/I344C (▾). Data are fitted as described in Materials and methods, giving K_d = 129 µM and nH = 1.36 for cys-less, K_d = 19.5 µM and nH = 1.21 for I344C, and K_d = 3.91 µM and nH = 1.65 for Q98C/I344C. (C) Mean K_d calculated from individual patches using fits of the kind shown in B. Asterisks indicate a significant difference from cys-less, and daggers indicate a significant difference between the double mutant indicated and either of the individual mutations alone (P < 0.05 in each case). Mean of data from four to seven patches shown in B and C.