Cysteine-scanning mutagenesis of transmembrane segment 11 of the GLUT1 facilitative glucose transporter

The glucose permeation pathway within the GLUT1 facilitative glucose transp orter is hypothesized to be formed by the juxtaposition of the hydrophilic faces of several transmembrane alpha-helices. The role of transmembrane seg ment 11 in forming a portion of this central aqueous channel was investigat ed using cysteine-scanning mutagenesis in conjunction with sulfhydryl-direc ted chemical modification. Each of the amino acid residues within transmemb rane segment 11 were individually mutated to cysteine in an engineered GLUT 1 molecule devoid of all native cysteines (C-less). Measurement of 2-deoxyg lucose uptake in a Xenopus oocyte expression system revealed that all of th ese mutants retain measurable transport activity. Four of the cysteine muta nts (N411, W412, N415, and F422) had significantly reduced specific activit y relative to the C-less protein. Specific activity was increased in five o f the mutants (A402, A405, V406, F416, and M420). The solvent accessibility and relative orientation of the residues to the glucose permeation pathway were investigated by determining the sensitivity of the mutant transporter s to inhibition by the sulfhydryl-directed reagent p-chloromercuribenzenesu lfonate (pCMBS). Cysteine replacement at five positions (I404, G408, F416, G419, and M420) produced transporters that were inhibited by incubation wit h extracellular pCMBS. All of these residues cluster along a single face of the alpha-helix within the regions showing altered specific activities. Th ese data demonstrate that the exofacial portion of transmembrane segment ii is accessible to the external solvent and provide evidence for the positio ning of this alpha-helix within or near the glucose permeation pathway.