Identification of a key residue determining substrate affinity in the human glucose transporter GLUT1

Asn³³¹ in transmembrane segment 7 of the yeast Saccharomyces cerevisiae transporter Hxt2 has been identified as a single key residue for high-affinity glucose transport by comprehensive chimera approach. The glucose transporter GLUT1 of mammals belongs to the same major facilitator superfamily as Hxt2 and may therefore show a similar mechanism of substrate recognition. The functional role of Ile²⁸⁷ in human GLUT1, which corresponds to Asn³³¹ in Hxt2, was studied by its replacement with each of the other 19 amino acids. The mutant transporters were individually expressed in a recently developed yeast expression system for GLUT1. Replacement of Ile²⁸⁷ generated transporters with various affinities for glucose that correlated well with those of the corresponding mutants of the yeast transporter. Residues exhibiting high affinity for glucose were medium-sized, non-aromatic, uncharged and irrelevant to hydrogen-bond capability, suggesting an important role of van der Waals interaction. Sensitivity to phloretin, a specific inhibitor for the presumed exofacial glucose binding site, was decreased in two mutants, whereas that to cytochalasin B, a specific inhibitor for the presumed endofacial glucose binding site, was unchanged in the mutants. These results suggest that Ile²⁸⁷ is a key residue for maintaining high glucose affinity in GLUT1 as revealed in Hxt2 and is located at or near the exofacial glucose binding site.