Ion permeation through a Cl−-selective channel designed from a CLC Cl−/H+ exchanger

The CLC family of Cl−-transporting proteins includes both Cl− channels and Cl−/H+ exchange transporters. CLC-ec1, a structurally known bacterial homolog of the transporter subclass, exchanges two Cl− ions per proton with strict, obligatory stoichiometry. Point mutations at two residues, Glu148 and Tyr445, are known to impair H+ movement while preserving Cl− transport. In the x-ray crystal structure of CLC-ec1, these residues form putative “gates” flanking an ion-binding region. In mutants with both of the gate-forming side chains reduced in size, H+ transport is abolished, and unitary Cl− transport rates are greatly increased, well above values expected for transporter mechanisms. Cl− transport rates increase as side-chain volume at these positions is decreased. The crystal structure of a doubly ungated mutant shows a narrow conduit traversing the entire protein transmembrane width. These characteristics suggest that Cl− flux through uncoupled, ungated CLC-ec1 occurs via a channel-like electrodiffusion mechanism rather than an alternating-exposure conformational cycle that has been rendered proton-independent by the gate mutations

Functional identification and characterization of sodium binding sites in Na symporters

Sodium cotransporters from several different gene families belong to the leucine transporter (LeuT) structural family. Although the identification of Na(+) in binding sites is beyond the resolution of the structures, two Na(+) binding sites (Na1 and Na2) have been proposed in LeuT. Na2 is conserved in the LeuT family but Na1 is not. A biophysical method has been used to measure sodium dissociation constants (K(d)) of wild-type and mutant human sodium glucose cotransport (hSGLT1) proteins to identify the Na(+) binding sites in hSGLT1. The Na1 site is formed by residues in the sugar binding pocket, and their mutation influences sodium binding to Na1 but not to Na2. For the canonical Na2 site formed by two –OH side chains, S392 and S393, and three backbone carbonyls, mutation of S392 to cysteine increased the sodium K(d) by sixfold. This was accompanied by a dramatic reduction in the apparent sugar and phlorizin affinities. We suggest that mutation of S392 in the Na2 site produces a structural rearrangement of the sugar binding pocket to disrupt both the binding of the second Na(+) and the binding of sugar. In contrast, the S393 mutations produce no significant changes in sodium, sugar, and phlorizin affinities. We conclude that the Na2 site is conserved in hSGLT1, the side chain of S392 and the backbone carbonyl of S393 are important in the first Na(+) binding, and that Na(+) binding to Na2 promotes binding to Na1 and also sugar binding