Transmembrane segment 6 of the Glut1 glucose transporter is an outer helix and contains amino acid side chains essential for transport activity

Abstract

Experimental data and homology modeling suggest a structure for the exofacial configuration of the Glut1 glucose transporter in which 8 transmembrane helices form an aqueous cavity in the bilayer that is stabilized by four outer helices. The role of transmembrane segment 6, predicted to be an outer helix in this model, was examined by cysteine-scanning mutagenesis and the substituted cysteine accessibility method using the membrane-impermeant, sulfhydryl-specific reagent, p-chloromercuribenzene-sulfonate (pCMBS). A fully functional Glut1 molecule lacking all 6 native cysteine residues was used as a template to produce a series of 21 Glut1 point mutants in which each residue along helix 6 was individually changed to cysteine. These mutants were expressed in Xenopus oocytes, and their expression levels, functional activities, and sensitivities to inhibition by pCMBS were determined. Cysteine substitutions at Leu(204) and Pro(205) abolished transport activity, whereas substitutions at Ile(192), Pro(196), Gln(200), and Gly(201) resulted in inhibition of activity that ranged from approximately 35 to approximately 80%. Cysteine substitutions at Leu(188), Ser(191), and Leu(199) moderately augmented specific transport activity relative to the control. These results were dramatically different from those previously reported for helix 12, the structural cognate of helix 6 in the pseudo-symmetrical structural model, for which none of the 21 single-cysteine mutants exhibited reduced activity. Only the substitution at Leu(188) conferred inhibition by pCMBS, suggesting that most of helix 6 is not exposed to the external solvent, consistent with its proposed role as an outer helix. These data suggest that helix 6 contains amino acid side chains that are critical for transport activity and that structurally analogous outer helices may play distinct roles in the function of membrane transporters.

FIGURE 1.

Expression of helix 6 single-C mutant transporters in Xenopus oocytes. Stage 5 Xenopus oocytes were injected with 50 ng of wild-type, C-less, or mutant C-less mRNAs, and 2 days later, frozen sections were prepared and analyzed by indirect immunofluorescence laser confocal microscopy or oocytes were used to prepare purified membrane fractions for immunoblot analysis. a, confocal micrographs of oocytes expressing each of the 21 single-C mutants. Sham, sham-injected oocytes. b, immunoblot. 10 μg of total oocyte membrane protein were loaded per lane. Rabbit antiserum A674 raised against the C-terminal 15 residues of human Glut1 was used at 1:500 dilution. Numbers above the lanes on the right represent the amount in ng of human erythrocyte Glut1 loaded in each lane as quantitative standards.

FIGURE 2.

2-Deoxyglucose (2-DOG) uptake activity of helix 6 single-C mutants. [³H]2-Deoxyglucose uptake (50 μm, 30 min at 22 °C) and the plasma membrane content of each single-C mutant were quantitated 2 days after injection of mRNAs. Results represent the mean ± S.E. of 6–8 independent experiments, each experiment employing 15–20 oocytes per experimental group. a, raw uptake data (^*, p < 0.01 for single-C mutants compared with parental C-less Glut1); b, the data were normalized per ng of each mutant protein expressed per 10 μg of total oocyte membrane (^*, p < 0.05 for single-C mutants compared with parental C-less Glut1). Background values observed in sham-injected oocytes were subtracted prior to normalization.

FIGURE 3.

Effect of pCMBS on transport activity of helix 6 single-C mutants. 3 days after injection of mRNAs, groups of 15–20 oocytes were incubated in the presence or absence of 0.5 mm pCMBS in Barth's saline at 22 °C for 15 min. Oocytes were washed four times in Barth's saline and then subjected to 2-deoxyglucose (2-DOG) uptake measurements under the conditions described in the legend for Fig. 2. Results represent the mean ± S.E. of 7–8 independent experiments, each experiment employing 15–20 oocytes per experimental group. Data are expressed as relative uptake activity, i.e. uptake observed in the presence of pCMBS divided by the uptake observed in the absence of pCMBS. C-less represents the parental cysteine-less Glut1 construct. V165C is a well characterized positive control whose activity is inhibited by pCMBS (32). ^*, p < 0.01 activity with versus without prior incubation in the presence of pCMBS. ND, not determined.

FIGURE 4.

Helical wheel representation of helix 6. Transmembrane helix 6 of Glut1 as viewed from the exoplasmic surface of the membrane. Amino acids are represented by the single letter code. The black arrows point to the residues where cysteine substitution resulted in an inhibition of transport activity, and the red arrow points to the single residue that is exposed to the external solvent according to pCMBS reactivity.

FIGURE 5.

Low resolution model for the arrangement of the 12 transmembrane helices of Glut1. A proposed model of the exofacial glucose-binding site as viewed from the outside of the cell is shown. For simplicity, all transmembrane segments are drawn as perfect helices perpendicular to the plane of the membrane. Glucose is not drawn to scale. The dotted lines represent possible hydrogen bonds formed between glucose hydroxyl groups and various side chains on Glut1. Numbered residues are accessible to pCMBS from the external solvent. Helix 9 is shown in gray because it has not yet been analyzed by scanning mutagenesis.