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

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.