title: Synergism between halide binding and proton transport in a CLC-type exchanger.
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.
authors.affiliation: Department of Biochemistry, Howard Hughes Medical Institute, Brandeis University, Waltham, MA 02454-9110, USA.
authors.forename: Alessio
authors.initials: A
authors.lastname: Accardi
authors.forename: Séverine
authors.initials: S
authors.lastname: Lobet
authors.forename: Carole
authors.initials: C
authors.lastname: Williams
authors.forename: Christopher
authors.initials: C
authors.lastname: Miller
authors.forename: Raimund
authors.initials: R
authors.lastname: Dutzler
mesh_terms: D001965:Bromides / Q000378:metabolism*; D018118:Chloride Channels / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D017136:Ion Transport / Q000502:physiology*; D050505:Mutant Proteins / Q000378:metabolism; D017433:Protein Structure, Secondary; D011522:Protons
chemical_list: D001965:Bromides; D018118:Chloride Channels; C508370:ClC protein, E coli; D029968:Escherichia coli Proteins; D050505:Mutant Proteins; D011522:Protons