Cysteine-scanning mutagenesis of transmembrane segment 7 of the GLUT1 glucose transporter

The human erythrocyte facilitative glucose transporter (Glut1) is predicted to contain 12 transmembrane spanning alpha-helices based upon hydropathy p lot analysis of the primary sequence. Five of these helices (3, 5, 7, 8, an d 11) are capable of forming amphipathic structures, A model of GLUT1 terti ary structure has therefore been proposed in which the hydrophilic faces of several amphipathic helices are arranged to form a central aqueous channel through which glucose traverses the hydrophobic lipid bilayer. In order to test this model, we individually mutated each of the amino acid residues i n transmembrane segment 7 to cysteine in an engineered GLUT1 molecule devoi d of all native cysteines (C-less). Measurement of 2-deoxyglucose uptake in a Xenopus oocyte expression system revealed that nearly all of these mutan ts retain measurable transport activity, Over one-half of the cysteine muta nts had significantly reduced specific activity relative to the C-less prot ein. The solvent accessibility and relative orientation of the residues wit hin the helix was investigated by determining the sensitivity of the mutant transporters to inhibition by the sulfhydryl directed reagent p-chloromerc uribenzene sulfonate (pCMBS). Cysteine replacement at six positions (GLn(28 2), Gln(283), Ile(287), Ala(289), Val(290), and Phe(291)), all near the exo facial side of the cell membrane, produced transporters that were inhibited by incubation with extracellular pCMBS. Residues predicted to be near the cytoplasmic side of the cell membrane were minimally affected by pCMBS. The se data demonstrate that the exofacial portion of transmembrane segment 7 i s accessible to the external solvent and provide evidence for the positioni ng of this alpha-helix within the glucose permeation pathway.