Department of Anesthesiology

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Synergism between halide binding and proton transport in a CLC-type exchanger.

TitleSynergism between halide binding and proton transport in a CLC-type exchanger.
Publication TypeJournal Article
Year of Publication2006
AuthorsAccardi A, Lobet S, Williams C, Miller C, Dutzler R
JournalJ Mol Biol
Volume362
Issue4
Pagination691-9
Date Published2006 Sep 29
ISSN0022-2836
KeywordsBromides, Chloride Channels, Escherichia coli, Escherichia coli Proteins, Ion Transport, Mutant Proteins, Protein Structure, Secondary, Protons
Abstract

The Cl-/H+ exchange-transporter CLC-ec1 mediates stoichiometric transmembrane exchange of two Cl- ions for one proton. A conserved tyrosine residue, Y445, coordinates one of the bound Cl- ions visible in the structure of this protein and is located near the intersection of the Cl- and H+ pathways. Mutants of this tyrosine were scrutinized for effects on the coupled transport of Cl- and H+ determined electrophysiologically and on protein structure determined crystallographically. Despite the strong conservation of Y445 in the CLC family, substitution of F or W at this position preserves wild-type transport behavior. Substitution by A, E, or H, however, produces uncoupled proteins with robust Cl- transport but greatly impaired movement of H+. The obligatory 2 Cl-/1 H+ stoichiometry is thus lost in these mutants. The structures of all the mutants are essentially identical to wild-type, but apparent anion occupancy in the Cl- binding region correlates with functional H+ coupling. In particular, as determined by anomalous diffraction in crystals grown in Br-, an electrophysiologically competent Cl- analogue, the well-coupled transporters show strong Br- electron density at the "inner" and "central" Cl- binding sites. However, in the uncoupled mutants, Br- density is absent at the central site, while still present at the inner site. An additional mutant, Y445L, is intermediate in both functional and structural features. This mutant clearly exchanges H+ for Cl-, but at a reduced H+-to-Cl- ratio; likewise, both the central and inner sites are occupied by Br-, but the central site shows lower Br- density than in wild-type (or in Y445F,W). The correlation between proton coupling and central-site occupancy argues that halide binding to the central transport site somehow facilitates movement of H+, a synergism that is not readily understood in terms of alternating-site antiport schemes.

DOI10.1016/j.jmb.2006.07.081
Alternate JournalJ. Mol. Biol.
PubMed ID16949616
Grant ListGM-31768 / GM / NIGMS NIH HHS / United States