Uncoupling and Turnover in a Cl−/H+ Exchange Transporter

Abstract

The CLC-family protein CLC-ec1, a bacterial homologue of known structure, stoichiometrically exchanges two Cl⁻ for one H⁺ via an unknown membrane transport mechanism. This study examines mutations at a conserved tyrosine residue, Y445, that directly coordinates a Cl⁻ ion located near the center of the membrane. Mutations at this position lead to “uncoupling,” such that the H⁺/Cl⁻ transport ratio decreases roughly with the volume of the substituted side chain. The uncoupled proteins are still able to pump protons uphill when driven by a Cl⁻ gradient, but the extent and rate of this H⁺ pumping is weaker in the more uncoupled variants. Uncoupling is accompanied by conductive Cl⁻ transport that is not linked to counter-movement of H⁺, i.e., a “leak.” The unitary Cl⁻ transport rate, measured in reconstituted liposomes by both a conventional initial-velocity method and a novel Poisson dilution approach, is ∼4,000 s⁻¹ for wild-type protein, and the uncoupled mutants transport Cl⁻ at similar rates.