title: Separate ion pathways in a Cl-/H+ exchanger.
abstract: CLC-ec1 is a prokaryotic CLC-type Cl(-)/H+ exchange transporter. Little is known about the mechanism of H+ coupling to Cl-. A critical glutamate residue, E148, was previously shown to be required for Cl(-)/H+ exchange by mediating proton transfer between the protein and the extracellular solution. To test whether an analogous H+ acceptor exists near the intracellular side of the protein, we performed a mutagenesis scan of inward-facing carboxyl-bearing residues and identified E203 as the unique residue whose neutralization abolishes H+ coupling to Cl- transport. Glutamate at this position is strictly conserved in all known CLCs of the transporter subclass, while valine is always found here in CLC channels. The x-ray crystal structure of the E203Q mutant is similar to that of the wild-type protein. Cl- transport rate in E203Q is inhibited at neutral pH, and the double mutant, E148A/E203Q, shows maximal Cl- transport, independent of pH, as does the single mutant E148A. The results argue that substrate exchange by CLC-ec1 involves two separate but partially overlapping permeation pathways, one for Cl- and one for H+. These pathways are congruent from the protein's extracellular surface to E148, and they diverge beyond this point toward the intracellular side. This picture demands a transport mechanism fundamentally different from familiar alternating-access schemes.
authors.affiliation: Department of Biochemistry, Howard Hughes Medical Institute, Brandeis University, Waltham, MA 02454, USA.
authors.forename: Alessio
authors.initials: A
authors.lastname: Accardi
authors.forename: Michael
authors.initials: M
authors.lastname: Walden
authors.forename: Wang
authors.initials: W
authors.lastname: Nguitragool
authors.forename: Hariharan
authors.initials: H
authors.lastname: Jayaram
authors.forename: Carole
authors.initials: C
authors.lastname: Williams
authors.forename: Christopher
authors.initials: C
authors.lastname: Miller
mesh_terms: D000595:Amino Acid Sequence; D018118:Chloride Channels / Q000737:chemistry* / Q000378:metabolism; D002712:Chlorides / Q000378:metabolism; D003461:Crystallography; D004926:Escherichia coli / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D017136:Ion Transport; D008969:Molecular Sequence Data; D009154:Mutation; D011487:Protein Conformation; D017494:Proton Pumps / Q000737:chemistry* / Q000378:metabolism
chemical_list: D018118:Chloride Channels; D002712:Chlorides; D017494:Proton Pumps