Paradoxical [ATP] dependence of G551D-CFTR currents supports the hypothesis of two ATP-binding sites exerting opposite actions. (A) Reversed ATP-dose dependence in G551D-CFTR. A switch from 2 mM to 20 µM ATP increased the current of G551D-CFTR, and the effect readily reversed once the ATP concentration was increased. The current in the presence of 20 µM ATP was 1.66-fold higher compared with that of 2 mM ATP (P < 0.05; paired t test). The current can be inhibited by a specific CFTR inhibitor, Inh-172 (Kopeikin et al., 2010). Note a biphasic current change is also seen when ATP removal takes place concurrently with the addition of Inh-172. This is likely because of a fast relief of ATP-dependent inhibition but a slow onset of current inhibition by Inh-172. Of note, an additional kinetic step is required after binding of Inh-172 to inhibit CFTR gating (Kopeikin et al., 2010). (B) A continuous recording of G551D-CFTR showing bell-shaped [ATP] dependence. Note a current increase when [ATP] is decreased from 2 mM to 20 µM, but a decrease of the current when [ATP] is further reduced to 1 µM. (C) Inverse [ATP] dependence of G551D-CFTR currents in the absence of VX-770. Note a reversible increase of the current upon switching [ATP] from 2 mM to 20 µM. The current in the presence of 20 µM ATP was 1.34-fold higher than that of 2 mM ATP (P < 0.05, paired t test). (D) Fold increase of G551D-CFTR currents upon switching solution from 2 mM to 20 µM ATP (I_20µM/I_2mM) in the presence or absence of VX-770. Mean ± SEM is shown.