Synergism Between Halide Binding and Proton Transport in a CLC-type Exchanger

The Cl−/H+ exchange-transporter CLC-ec1 mediates stoichiometric transmembrane exchange of two Cl− ions for one proton. A conserved tyrosine residue, Y445, coordinates one of the bound Cl− ions visible in the structure of this protein and is located near the intersection of the Cl− and H+ pathways. Mutants of this tyrosine were scrutinized for effects on the coupled transport of Cl− and H+ determined electrophysiologically and on protein structure determined crystallographically. Despite the strong conservation of Y445 in the CLC family, substitution of F or W at this position preserves wild-type transport behavior. Substitution by A, E, or H, however, produces uncoupled proteins with robust Cl− transport but greatly impaired movement of H+. The obligatory 2 Cl−/1 H+ stoichiometry is thus lost in these mutants. The structures of all the mutants are essentially identical to wild-type, but apparent anion occupancy in the Cl− binding region correlates with functional H+ coupling. In particular, as determined by anomalous diffraction in crystals grown in Br−, an electrophysiologically competent Cl− analogue, the well-coupled transporters show strong Br− electron density at the “inner” and “central” Cl− binding sites. However, in the uncoupled mutants, Br− density is absent at the central site, while still present at the inner site. An additional mutant, Y445L, is intermediate in both functional and structural features. This mutant clearly exchanges H+ for Cl−, but at a reduced H+-to-Cl− ratio; likewise, both the central and inner sites are occupied by Br−, but the central site shows lower Br− density than in wild-type (or in Y445F,W). The correlation between proton coupling and central-site occupancy argues that halide binding to the central transport site somehow facilitates movement of H+, a synergism that is not readily understood in terms of alternating-site antiport schemes.