Comparisons of the modification rate constant in the presence or absence of ATP suggest that there is no cytoplasmic-side gate in the CFTR channel. (A) A representative experiment for measuring the modification rate in the absence of ATP. Macroscopic CFTR currents elicited by applications (black arrows) of 2 mM ATP were gradually decreased upon each short application (red arrows) of MTSES⁻ until the current level reached a steady state. (B and C) The ordinate shows the normalized current decay induced by MTSES⁻ modification in the presence (continuous traces) or absence (closed squares) of ATP fitted into single-exponential functions (red lines). The abscissa shows accumulated MTSES⁻ exposure (the concentration of MTSES− times the duration of the application). Note that the modification is faster in the presence of ATP than in the absence of ATP for cysless/N1148C-CFTR channels (B), whereas it is slower in the presence of ATP than in the absence of ATP for cysless/S1141C-CFTR channels (C). (D) Summary of second-order modification rate constants (κ_MTSES⁻) with (open circles) or without (closed squares) ATP on a logarithmic scale. Black arrows connecting the two symbols indicate that κ_MTSES⁻ with ATP is larger than that without ATP, whereas gray arrows indicate that κ_MTSES⁻ with ATP is smaller than that without ATP. Crossed circles mark cysteine-substituted channels whose function was not significantly altered by the treatment of MTSES⁻ either with or without ATP. Each data point is the average of four to seven patches. Symbols are larger than SEM.