Department of Anesthesiology

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Uncoupling and turnover in a Cl-/H+ exchange transporter.

TitleUncoupling and turnover in a Cl-/H+ exchange transporter.
Publication TypeJournal Article
Year of Publication2007
AuthorsWalden M, Accardi A, Wu F, Xu C, Williams C, Miller C
JournalJ Gen Physiol
Volume129
Issue4
Pagination317-29
Date Published2007 Apr
ISSN0022-1295
KeywordsAmino Acid Substitution, Antiporters, Chloride Channels, Chlorides, Dimerization, Escherichia coli, Escherichia coli Proteins, Liposomes, Models, Chemical, Molecular Sequence Data, Mutation, Poisson Distribution, Protein Structure, Tertiary, Protons
Abstract

The CLC-family protein CLC-ec1, a bacterial homologue of known structure, stoichiometrically exchanges two Cl(-) for one H(+) via an unknown membrane transport mechanism. This study examines mutations at a conserved tyrosine residue, Y445, that directly coordinates a Cl(-) ion located near the center of the membrane. Mutations at this position lead to "uncoupling," such that the H(+)/Cl(-) transport ratio decreases roughly with the volume of the substituted side chain. The uncoupled proteins are still able to pump protons uphill when driven by a Cl(-) gradient, but the extent and rate of this H(+) pumping is weaker in the more uncoupled variants. Uncoupling is accompanied by conductive Cl(-) transport that is not linked to counter-movement of H(+), i.e., a "leak." The unitary Cl(-) transport rate, measured in reconstituted liposomes by both a conventional initial-velocity method and a novel Poisson dilution approach, is approximately 4,000 s(-1) for wild-type protein, and the uncoupled mutants transport Cl(-) at similar rates.

DOI10.1085/jgp.200709756
Alternate JournalJ. Gen. Physiol.
PubMed ID17389248
PubMed Central IDPMC2151619
Grant ListR01-31768 / / PHS HHS / United States