Direct Sensing of Intracellular pH by the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Cl− Channel

Jeng-Haur Chen,Zhiwei Cai,David N Sheppard

doi:10.1074/jbc.m109.072678

Abstract

In cystic fibrosis (CF), dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel disrupts epithelial ion transport and perturbs the regulation of intracellular pH (pH(i)). CFTR modulates pH(i) through its role as an ion channel and by regulating transport proteins. However, it is unknown how CFTR senses pH(i). Here, we investigate the direct effects of pH(i) on recombinant CFTR using excised membrane patches. By altering channel gating, acidic pH(i) increased the open probability (P(o)) of wild-type CFTR, whereas alkaline pH(i) decreased P(o) and inhibited Cl(-) flow through the channel. Acidic pH(i) potentiated the MgATP dependence of wild-type CFTR by increasing MgATP affinity and enhancing channel activity, whereas alkaline pH(i) inhibited the MgATP dependence of wild-type CFTR by decreasing channel activity. Because these data suggest that pH(i) modulates the interaction of MgATP with the nucleotide-binding domains (NBDs) of CFTR, we examined the pH(i) dependence of site-directed mutations in the two ATP-binding sites of CFTR that are located at the NBD1:NBD2 dimer interface (site 1: K464A-, D572N-, and G1349D-CFTR; site 2: G551D-, K1250M-, and D1370N-CFTR). Site 2 mutants, but not site 1 mutants, perturbed both potentiation by acidic pH(i) and inhibition by alkaline pH(i), suggesting that site 2 is a critical determinant of the pH(i) sensitivity of CFTR. The effects of pH(i) also suggest that site 2 might employ substrate-assisted catalysis to ensure that ATP hydrolysis follows NBD dimerization. We conclude that the CFTR Cl(-) channel senses directly pH(i). The direct regulation of CFTR by pH(i) has important implications for the regulation of epithelial ion transport.

Highlights

CFTR is assembled from five domains: two membrane-spanning domains (MSDs) that form an anion-selective pore, two nucleotide-binding domains (NBDs) that bind and hydrolyze
Acidic pHi potentiated the MgATP dependence of wild-type CFTR by increasing MgATP affinity and enhancing channel activity, whereas alkaline pHi inhibited the MgATP dependence of wildtype CFTR by decreasing channel activity. Because these data suggest that pHi modulates the interaction of MgATP with the nucleotide-binding domains (NBDs) of CFTR, we examined the pHi dependence of site-directed mutations in the two ATPbinding sites of CFTR that are located at the NBD1:NBD2 dimer interface
Multiple Effects of pHi on CFTR Activity—To investigate whether pHi regulates directly the CFTR ClϪ channel, we studied CFTR ClϪ currents in membrane patches excised from C127 cells expressing wild-type human CFTR

Summary

Introduction

CFTR is assembled from five domains: two membrane-spanning domains (MSDs) that form an anion-selective pore, two nucleotide-binding domains (NBDs) that bind and hydrolyze. 6C and 8 and supplemental Fig. 3, B and C, demonstrate that the gating behavior of D572N- and D1370N-CFTR ClϪ channels at different pHi diverges from that of wild-type CFTR in several important respects.

Results

Conclusion