The cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl^– channel with complex regulation. Channel gating is controlled by two nucleotide-binding domains and an R (regulatory) domain located on the cytoplasmic side of the cell membrane. The location of these domains suggests that their function might be regulated by intracellular pH. To test this hypothesis, we investigated the effect of changing intracellular pH on the activity of CFTR.

In this study, we used the patch-clamp technique to investigate CFTR Cl^– channels in excised inside-out membrane patches from C127 cells stably expressing wild-type human CFTR (Lansdell et al. 2000). The pipette (external) solution contained 10 mM Cl^– at pH 7.3, whereas the bath (internal) solution contained 147 mM Cl^–. To ensure that the Cl^– concentration was identical, all bath solutions were first titrated to pH 7.3 (control) with HCl before adding either H₂SO₄ to decrease pH or Tris to increase pH. Voltage was clamped at -50 mV and the bath solution was maintained at 37 °C. To stimulate and sustain the activity of CFTR Cl^– channels, PKA (75 nM) and ATP (0.3-1 mM) were added to all internal solutions. We expressed data as means ± S.E.M. of n observations and performed statistical analyses using Student’s paired t test.

We began by investigating the effects of changing intracellular pH on CFTR Cl^– currents. When the internal solution was acidified, the magnitude of CFTR Cl^– current increased (pH 7.3, 100% pH 6.3, 141 ± 9% n = 6; P < 0.001). In contrast, when the internal solution was alkalinised, the magnitude of CFTR Cl^– current decreased (pH 7.3, 100% pH 8.3, 70 ± 9% n = 6; P < 0.001). The effects of acid and alkaline pH on CFTR Cl^– currents were reversible. To investigate how intracellular pH regulates CFTR, we studied single CFTR Cl^– channels. At pH 6.3, the duration of channel openings was increased and the length of the long closures separating channel openings was decreased. As a result, at pH 6.3 the open probability (P_o) of CFTR was greatly increased (pH 7.3, P_o = 0.44 ± 0.02; pH 6.3, P_o = 0.62 ± 0.02; n = 5; P < 0.001). In contrast, at pH 8.3 the duration of channel openings decreased and the length of the long closures separating channel openings was increased. As a result, at pH 8.3, the P_o of CFTR was markedly decreased (pH 7.3, P_o = 0.42 ± 0.02; pH 8.3, P_o = 0.29 ± 0.03; n = 8; P < 0.001). Moreover, at pH 8.3 the single-channel current amplitude of CFTR was decreased (pH 7.3, -0.85 ± 0.02 pA; pH 8.3, -0.81 ± 0.02 pA; n = 8; P < 0.001). Acidifying the intracellular solution was without effect on i (n = 5). These data indicate that intracellular pH has multiple effects on the single-channel activity of CFTR. They also suggest that several protein regions within CFTR might contain pH-sensitive amino acid residues.

This work was supported by the CF Trust and the University of Bristol.