Time course of modification by MTSES and MTSET. (A) Example time courses of macroscopic currents (measured at −50 mV during brief voltage excursions from a holding potential of 0 mV) carried by K95C (left) and L102C (right) as indicated, in inside-out membrane patches. Current amplitudes were measured every 6 s after the attainment of stable current amplitude after channel activation with 20 nM PKA, 1 mM ATP, and 2 mM PPi. In each case, MTSES (20 µM for K95C and 200 µM for L102C) was applied to the cytoplasmic face of the patch at time zero (as indicated by the hatched bar at the bottom of each panel). The decline in current amplitude after MTSES application has been fitted by a single-exponential function in each case. (B) Calculated modification rate constants for both MTSES (○) and MTSET (•) for each of the four MTS reagent–sensitive mutants listed. Asterisks indicate a significant difference from MTSES modification of K95C (P < 0.005), and daggers indicate a significant difference from MTSES modification of the same mutant (P < 0.05). Mean of data from three patches in each case is shown.