Identification of positive charges situated at the outer mouth of the CFTR chloride channel pore |
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Authors: | Jing-Jun Zhou Mohammad Fatehi Paul Linsdell |
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Institution: | (1) Department of Physiology & Biophysics, Dalhousie University, 5850 College Street, Halifax, NS, B3H 1X5, Canada;(2) Present address: Department of Physiology, Fourth Military Medical University, #17 Changle Western Road, Xi’an, 710032, People’s Republic of China;(3) Present address: Department of Biomedical Sciences, Atlantic Veterinary College, University of Prince Edward Island, 550 University Avenue, Charlottetown, PE, C1A 4P3, Canada |
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Abstract: | 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. |
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Keywords: | Chloride channel Conductance Cystic fibrosis transmembrane conductance regulator Single channel Site-directed mutagenesis Surface charge |
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