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.