Evidence for the involvement of Ala 166 in coupling Na+ to sugar transportthrough the human Na+/glucose cotransporter

We mutated residue 166, located in the putative Na+ transport pathway betwe en transmembrane segments 4 and 5 of human Na+/glucose cotransporter (hSGLT 1), from alanine to cysteine (A166C). A166C was expressed in Xenopus laevis oocytes, and electrophysiological methods were used to assay function. The affinity for Na+ was unchanged compared to that of hSGLT1, whereas the sug ar affinity was reduced and sugar specificity was altered. There was a redu ction in the turnover rate of the transporter, and in contrast to that of h SGLT1, the turnover rate depended on the sugar molecule. Exposure of A166C to MTSEA and MTSET, but not MTSES, abolished sugar transport. Accessibility of A166C to alkylating reagents was independent of protein conformation, i ndicating that the residue is always accessible from the extracellular surf ace. Sugar and phlorizin did not protect the residue from being alkylated, suggesting that residue 166 is not located in the sugar pathway. MTSEA, MTS ET, and MTSES all changed the pre-steady-state kinetics of A166C, independe nt of pH, and sugars altered these kinetics. The inability of MTSEA-labeled A166C to transport sugar was reversed (with no major change in Na+ and sug ar affinity) if the positive charge on MTSEA was neutralized by increasing the external pH to 9.0. These studies suggest that the residue at position 166 is involved in the interaction between the Na+ and sugar transport path ways.