Separate ion pathways in a Cl-/H+ exchanger.
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.
Accardi
Alessio
A
Department of Biochemistry, Howard Hughes Medical Institute, Brandeis University, Waltham, MA 02454, USA.
Walden
Michael
M
Nguitragool
Wang
W
Jayaram
Hariharan
H
Williams
Carole
C
Miller
Christopher
C
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
D018118:Chloride Channels; D002712:Chlorides; D017494:Proton Pumps