title: Molecular determinants and role of an anion binding site in the external mouth of the CFTR chloride channel pore.
abstract: Chloride permeation through the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel is blocked by highly lyotropic permeant anions which bind tightly within the pore. Here we show that several different substitutions of a positively charged amino acid residue, arginine R334, in the putative outer mouth of the CFTR pore, greatly reduce the block caused by lyotropic Au(CN)2- ions applied to the intracellular side of the channel. Fixed positive charge at this site appears to play a role in Au(CN)2- binding, as judged by multiple substitutions of differently charged amino acid side chains and also by the pH dependence of block conferred by the R334H mutant. However, non-charge-dependent effects also appear to contribute to Au(CN)2- binding. Mutation of R334 also disrupts the apparent electrostatic interaction between intracellular Au(CN)2- ions and extracellular permeant anions, an interaction which normally acts to relieve channel block. All six mutations studied at R334 significantly weakened this interaction, suggesting that arginine possesses a unique ability to coordinate ion-ion interactions at this site in the pore. Our results suggest that lyotropic anions bind tightly to a site in the outer mouth of the CFTR pore that involves interaction with a fixed positive charge. Binding to this site is also involved in coordination of multiple permeant anions within the pore, suggesting that anion binding in the outer mouth of the pore is an important aspect in the normal anion permeation mechanism.
authors.affiliation: Department of Physiology and Biophysics, Dalhousie University, Halifax, Nova Scotia, Canada.
authors.forename: Xiandi
authors.initials: X
authors.lastname: Gong
authors.forename: Paul
authors.initials: P
authors.lastname: Linsdell
mesh_terms: D000818:Animals; D000838:Anions / Q000378:metabolism*; D001120:Arginine; D001665:Binding Sites; D006224:Cricetinae; D003485:Cyanates; D003486:Cyanides / Q000378:metabolism / Q000494:pharmacology; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D004594:Electrophysiology; D006046:Gold; D017612:Gold Compounds / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism; D015394:Molecular Structure; D009154:Mutation / Q000502:physiology
chemical_list: D000838:Anions; C505032:CFTR protein, human; D003485:Cyanates; D003486:Cyanides; D017612:Gold Compounds; C043041:gold cyanide; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D006046:Gold; D001120:Arginine