Department of Anesthesiology

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Separate ion pathways in a Cl-/H+ exchanger.

TitleSeparate ion pathways in a Cl-/H+ exchanger.
Publication TypeJournal Article
Year of Publication2005
AuthorsAccardi A, Walden M, Nguitragool W, Jayaram H, Williams C, Miller C
JournalJ Gen Physiol
Volume126
Issue6
Pagination563-70
Date Published2005 Dec
ISSN0022-1295
KeywordsAmino Acid Sequence, Chloride Channels, Chlorides, Crystallography, Escherichia coli, Hydrogen-Ion Concentration, Ion Transport, Molecular Sequence Data, Mutation, Protein Conformation, Proton Pumps
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.

DOI10.1085/jgp.200509417
Alternate JournalJ. Gen. Physiol.
PubMed ID16316975
PubMed Central IDPMC2266597
Grant ListGM-31768 / GM / NIGMS NIH HHS / United States