CONFORMATIONAL-CHANGES COUPLE NA-TRANSPORT( AND GLUCOSE)

The mechanism by which cotransport proteins couple their substrates ac ross cell membranes is not known, A commonly proposed model is that co transport results from ligand-induced conformational transitions that change the accessibility of ligand-binding sites from one side of the membrane to the other. To test this model, we have measured the access ibility of covalent probes to a cysteine residue (Q457C) placed in the putative sugar-translocation domain of the Na+/glucose cotransporter (SGLT1). The mutant protein Q457C was able to transport sugar, but tra nsport was abolished after alkylation by methanethiosulfonate reagents . Alkylation blocked sugar translocation but not sugar binding. Access ibility of Q457C to alkylating reagents required external Naf and was blocked by external sugar and phlorizin. The voltage dependence of acc essibility was directly correlated with the presteady-state charge mov ement of SGLT1. Voltage-jump experiments with rhodamine-6-maleimide-la beled Q457C showed that the time course and level of changes in fluore scence closely followed the presteady-state charge movement. We conclu de that conformational changes are responsible for the coupling of Naf and sugar transport and that Q457 plays a critical role in sugar tran slocation by SGLT1.