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.

CLC Cl − /H + transporters constrained by covalent cross-linking

“…1). The reversal potential (51 mV) of the ''strained'' dimer 207C-207C is slightly lower, but this value still represents respectably tight exchange coupling, especially when compared with variously uncoupled mutants previously described (11,15,16). We conclude, then, that the normal mechanism of Cl Ϫ /H ϩ transport is unperturbed in the covalent-dimer constructs.…”

“…The striking result of Fig. 7A is that all these CLC-ec1 variants give identical f o values and similar unitary transport rates, on the order of 2,000 s Ϫ1 , about half the rate of wild-type protein (11). Rates of the covalent dimers are 50-85% of the Cys-less rate, with the straitjacketed triple mutant being the slowest, 1,700 s Ϫ1 .…”

“…The portion of the efflux that requires detergent for release, labeled f o in Fig. 7A, reflects the protein-free liposomes and serves as a quantitative indicator of the fraction of protein that is functionally reconstituted (11,17). The striking result of Fig.…”

“…The reversal potential, a null-point measurement, tells us nothing about the absolute ion-transport rate catalyzed by the exchanger. To measure this rate, we followed passive Cl Ϫ efflux under Poisson dilution conditions, i.e., at protein concentrations so low that most of the transporting liposomes contain only a single copy of the transporter and the remaining liposomes have no protein at all (11). Reconstituted liposomes loaded with high Cl Ϫ were suspended in low-Cl Ϫ solution, and KCl efflux was followed under conditions in which Cl Ϫ transport is rate-limiting.…”

“…Moreover, the conserved glutamates corresponding to the fast gate in the channel subclass also are located in this Cl Ϫ -binding area and are crucially involved in coupling H ϩ to the movement of Cl Ϫ (1). In addition, the turnover rate of Cl Ϫ /H ϩ exchange (11) is in the same range as the kinetics of fast gating in the channels. On the other hand, coordinated countertransport of Cl Ϫ and H ϩ requires a cycle of conformational changes that must be more elaborate than rotation of a single glutamate sidechain; conformations should therefore exist that have not yet been seen in crystal structures, and these could in principle involve movements of the subunits with respect to each other.…”