title: Altering intracellular pH reveals the kinetic basis of intraburst gating in the CFTR Cl-channel.
abstract: The cystic fibrosis transmembrane conductance regulator (CFTR), which is defective in the genetic disease cystic fibrosis (CF), forms a gated pathway for chloride movement regulated by intracellular ATP. To understand better CFTR function, we investigated the regulation of channel openings by intracellular pH. We found that short-lived channel closures during channel openings represent subtle changes in the structure of CFTR that are regulated by intracellular pH, in part, at ATP-binding site 1 formed by the nucleotide-binding domains. Our results provide a framework for future studies to understand better the regulation of channel openings, the dysfunction of CFTR in CF and the action of drugs that repair CFTR gating defects.
  
  
  
  Cystic fibrosis transmembrane conductance regulator (CFTR) is an ATP-gated Cl-channel defective in the genetic disease cystic fibrosis (CF). The gating behaviour of CFTR is characterized by bursts of channel openings interrupted by brief, flickery closures, separated by long closures between bursts. Entry to and exit from an open burst is controlled by the interaction of ATP with two ATP-binding sites, sites 1 and 2, in CFTR. To understand better the kinetic basis of CFTR intraburst gating, we investigated the single-channel activity of human CFTR at different intracellular pH (pHi) values. When compared with the control (pHi7.3), acidifying pHito 6.3 or alkalinizing pHito 8.3 and 8.8 caused small reductions in the open-time constant (τo) of wild-type CFTR. By contrast, the fast closed-time constant (τcf), which describes the short-lived closures that interrupt open bursts, was greatly increased at pHi5.8 and 6.3. To analyse intraburst kinetics, we used linear three-state gating schemes. All data were satisfactorily modelled by the C1↔ O ↔ C2kinetic scheme. Changing the intracellular ATP concentration was without effect on τo, τcfand their responses to pHichanges. However, mutations that disrupt the interaction of ATP with ATP-binding site 1, including K464A, D572N and the CF-associated mutation G1349D all abolished the prolongation of τcfat pHi6.3. Taken together, our data suggest that the regulation of CFTR intraburst gating is distinct from the ATP-dependent mechanism that controls channel opening and closing. However, our data also suggest that ATP-binding site 1 modulates intraburst gating.
authors: School of Biomedical Sciences, University of Hong Kong, Hong Kong.|The University of Hong Kong Shenzhen Institute of Research and Innovation, Shenzhen, China.|School of Physiology, Pharmacology and Neuroscience, University of Bristol, Bristol, UK.
authors: Jeng-Haur
authors: 0000-0001-6756-6939
authors: JH
authors: Chen
authors: School of Biomedical Sciences, University of Hong Kong, Hong Kong.|The University of Hong Kong Shenzhen Institute of Research and Innovation, Shenzhen, China.
authors: Weiyi
authors: W
authors: Xu
authors: School of Physiology, Pharmacology and Neuroscience, University of Bristol, Bristol, UK.
authors: David N
authors: 0000-0001-5533-9130
authors: DN
authors: Sheppard
mesh_terms: D016475:3T3 Cells; D000200:Action Potentials; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000737:chemistry / Q000378:metabolism*; D005110:Extracellular Space / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D015640:Ion Channel Gating; D051379:Mice; D011485:Protein Binding; D051381:Rats
chemical_list: C505032:CFTR protein, human; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D000255:Adenosine Triphosphate
keywords: chloride channel; cystic fibrosis; cystic fibrosis transmembrane conductance regulator