The cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl^–channel with complex regulation. Channel gating is controlled by phosphorylation of the R domain and ATP binding and hydrolysis at two nucleotide-binding domains (NBDs). We previously demonstrated that intracellular pH (pH_i) regulates CFTR channel gating with acidic pH increasing open probability (P_o), but alkaline pH decreasing P_o and single-channel current amplitude (Chen et al. 2002). To understand better how pH_i regulates channel gating, we investigated the CFTR mutant CFTR∆R-S660A and the ATP-dependence of wild-type CFTR.We studied CFTR Cl^–channels in excised inside-out membrane patches from C127 cells expressing wild-type or mutant CFTR. The pipette (external) solution contained 10 mM Cl^–at pH 7.3. The bath (internal) solution contained 147 mM Cl^–,PKA (75 nM) and ATP (0.3 mM) at 37 °C. To adjust the bath solution to pH 8.3 and pH 6.3, we used Tris and H₂SO₄,respectively.To investigate the role of the R domain, we studied CFTR∆R-S660A, a mutant that deletes much of the R domain (residues 708-835) abolishing the PKA-dependence of CFTR. In contrast to wild-type CFTR, the P_o of CFTR∆R-S660A was low at both pH 7.3 and 8.3 (Student′s paired t-test; p = 0.36). However, acidic pH_i stimulated greatly CFTR∆R-S660A (pH 7.3, P_o = 0.06 ± 0.01; pH 6.3, P_o = 0.26 ± 0.06; means ± SEM; n = 6; p < 0.05). These data suggest that the R domain might, in part, mediate the inhibitory, but not the stimulatory, effects of pH_i on CFTR channel gating.To examine the role of the NBDs, we studied the effects of [MgATP]_i (0-10 mM, n = 6-7) on wild-type CFTR channel gating at different pH_i.The relationship between P and [MgATP]_i was best fit by Michaelis-Menten o functions at each pH_i.However, at pH 6.3, the K_m value was much reduced compared with those at pH 7.3 and 8.3 (pH 6.3, K_m = 36 µM; pH 7.3, K_m = 90 µM; pH 8.3, K_m = 101 µM). Moreover, the maximum P_o (P_omax) was increased at pH 6.3, but decreased at pH 8.3 (pH 6.3,P_omax = 0.71; pH 7.3, P_omax = 0.61; pH 8.3, P_omax = 0.49). These data suggest that pH_i might alter both the ATP affinity and ATPase activity of the NBDs. To understand better the effects of pH_i on the NBDs, we used Mg²⁺-free bath solutions containing ATP (3 mM). In the absence of Mg²⁺,P_o was greatly decreased at pH 7.3 and 8.3, but less reduced at pH 6.3 (n = 5-7). These data suggest several conclusions. First, acidic pH might enhance channel gating by increasing ATP binding to the NBDs. Second, alkaline pH might inhibit channel gating by slowing ATP hydrolysis at the NBDs. Third, amino acid residues in the NBDs might account for most of the pH_i dependence of CFTR channel gating.