Identification of positive charges situated at the outer mouth of the CFTR chloride channel pore.
We have used site-directed mutagenesis and functional analysis to identify positively charged amino acid residues in the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel that interact with extracellular anions. Mutation of two positively charged arginine residues in the first extracellular loop (ECL) of CFTR, R104, and R117, as well as lysine residue K335 in the sixth transmembrane region, leads to inward rectification of the current-voltage relationship and decreased single channel conductance. These effects are dependent on the charge of the substituted side chain and on the Cl(-) concentration, suggesting that these positive charges normally act to concentrate extracellular Cl(-) ions near the outer mouth of the pore. Side chain charge-dependent effects are mimicked by manipulating charge in situ by mutating these amino acids to cysteine followed by covalent modification with charged cysteine-reactive reagents, confirming the location of these side chains within the pore outer vestibule. State-independent modification of R104C and R117C suggests that these residues are located at the outermost part of the pore. We suggest that ECL1 contributes to the CFTR pore external vestibule and that positively charged amino acid side chains in this region act to attract Cl(-) ions into the pore. In contrast, we find no evidence that fixed positive charges in other ECLs contribute to the permeation properties of the pore.
Zhou
Jing-Jun
JJ
Department of Physiology & Biophysics, Dalhousie University, Halifax, NS, B3H 1X5, Canada.
Fatehi
Mohammad
M
Linsdell
Paul
P
D000818:Animals; D001120:Arginine; D002460:Cell Line; D002712:Chlorides / Q000378:metabolism*; D006224:Cricetinae; D003545:Cysteine; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008239:Lysine; D008564:Membrane Potentials; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010539:Permeability; D017671:Platinum Compounds / Q000494:pharmacology; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D055672:Static Electricity; D013329:Structure-Activity Relationship; D014162:Transfection
D002712:Chlorides; D017671:Platinum Compounds; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D001120:Arginine; D008239:Lysine; D003545:Cysteine