Effects of 3NB and 3PP on gating rates under nonhydrolytic conditions. (A and B) Macroscopic K1250A CFTR currents at −20 mV, elicited by exposures to 10 mM ATP (gray bars) in the absence of drug or in the presence of either 32 mM 3NB (A, blue bar) or 20 mM 3PP (B, red bar). All current decay time courses after ATP removal were fitted by single exponentials (colored lines), and time constants are indicated. (C) Macroscopic closing rates (bars; 1/τ; see Materials and methods) in the absence (gray bar) or presence of 32 mM 3NB (blue bar) or 20 mM 3PP (red bar) quantify effects on rate k−1 (cartoon, purple arrow). The K1250A mutation (cartoon, red stars) disrupts ATP hydrolysis in site 2 (red cross). (D and E) Macroscopic K1250A CFTR currents elicited by 10 mM ATP at −20 mV and prolonged exposures to 32 mM 3NB (D, blue bars) or 20 mM 3PP (E, red bars) of channels gating at steady-state. Zero-current levels (dotted lines) were estimated from final segments. In D, brief exposure to 3NB of surviving locked-open channels after ATP removal (20-s yellow box, expanded in inset) measures fractional pore block (see Fig. 3). (F) Fractional K1250A CFTR currents at −20 mV in 32 mM 3NB (left pair of bars) or 20 mM 3PP (right pair of bars) applied during steady-state gating (gray bars) or in the locked-open state (yellow bars). (G) Effects of 3NB (blue bar) and 3PP (red bar) on the closed-open equilibrium (cartoon, purple double arrow). Fractional effects on Po for K1250A CFTR were calculated as in Fig. 5 (D and H). Mean ± SEM is shown.
