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.