Insensitivity to CuPhe before channel activation. (A and B) Example leak-subtracted I-V relationships for K95C/I344C (A) and Q98C/I344C (B) after channel activation with 20 nM PKA and 1 mM ATP in inside-out patches that had been pretreated with CuPhe for 2 min, and then washed to remove CuPhe. Currents were recorded before (control) and after (+DTT) the addition of 5 mM DTT to the cytoplasmic solution. (C) Mean effect of DTT on current amplitude in patches that had never seen CuPhe (no pretreatment), had been pretreated as in A and B (pretreatment CuPhe), or had been treated with CuPhe after channel activation (pretreatment CuPhe, PKA, ATP). In these experiments, an increase in current amplitude after DTT application is taken as evidence that disulfide bond formation had taken place. Asterisks indicate a significant difference from no pretreatment conditions (P < 0.05). Mean of data from three to six patches is shown in C.