Structural signatures and membrane helix 4 in GLUT1: inferences from human blood-brain glucose transport mutants

Abstract

Exon IV of SLC2A1, a multiple facilitator superfamily (MFS) transporter gene, is particularly susceptible to mutations that cause GLUT1 deficiency syndrome, a human encephalopathy that results from decreased glucose flux through the blood-brain barrier. Genotyping of 100 patients revealed that in a third of them who harbor missense mutations in the GLUT1 transporter, transmembrane domain 4 (TM4), encoded by SLC2A1 exon IV, contains mutant residues that have the periodicity of one face of a kinked alpha-helix. Arg-126, located at the amino terminus of TM4, is the locus for most of the mutations followed by other arginine and glycine residues located elsewhere in the transporter but conserved among MFS proteins. The Arg-126 mutants were constructed and assayed for protein expression, targeting, and transport capacity in Xenopus oocytes. The role of charge at position 126, as well as its accessibility, was investigated in R126H by determining its activity as a function of extracellular pH. The results indicate that intracellular charges at the MFS TM2-3 and TM8-9 signature loops and flanking TMs 3, 5, and 6 are critical for the structure of GLUT1 as are TM glycines and that TM4, located at the catalytic core of MFS proteins, forms a helix that surfaces into the extracellular solution where another proton facilitates transport.

Figures

FIGURE 1.

GLUT1 topological model. GLUT1 is represented in the context of the plasma membrane (gray rectangle) and comprising cytoplasmic amino- (left side) and carboxyl-terminal (right side) domains. The extracellular side of the membrane is situated on the top part of the schematic. The relative length of TMs follows the crystal structure of LacY, and the length of the extramembranous amino and carboxyl termini and interhelical loops is also drawn approximately to scale. Numbered circles indicating residue number identify the location of missense mutations.

FIGURE 2.

Glucose influx into human RBC. Zero-trans velocity of influx of glucose into normal and patient RBCs was normalized to normal (control) maximum velocity (Vmax) obtained from Lineweaver-Burke plots of uptake and expressed as relative uptake velocity as a function of extracellular glucose concentration. The lower set of symbols (open symbols and crosses) represents uptake measured in seven normal samples. The fitted line represents a fitted Lineweaver-Burke relationship using a larger set of normal samples (n = 30). Filled symbols reflect uptake by mutant RBCs associated with complete loss of function of one allele (▪, deletion of nucleotides 616–617 causing a frameshift; ▴, R330X, a truncating mutation; ♦, R126C, a missense mutant). Three fitted lines (upper set) indicate Lineweaver-Burke relationships for these mutants. Gray diamonds (♦) indicate transport by G130S; data are fit by a similar linear relationship.

FIGURE 3.

Expression and targeting of GLUT1 and Arg-126 mutants in oocytes. A, total normal human RBC membranes (RBC, leftmost lane) and oocyte membrane extracts after reaction with the same antibodies. Additional water-injected oocyte and purified commercial GLUT1 controls were added and labeled as in B. B, purified oocyte plasma membrane-enriched extract stained with GLUT1 antibody. Water-injected oocyte membranes (Ø, leftmost lane) and purified GLUT1 (Control, rightmost lane, 45–49 kDa, obtained from FabGennix, Inc.) were added in parallel as controls to GLUT1-injected (wild type (WT)) and mutant-injected oocyte membranes. C, confocal microscopy of oocyte sections using GLUT1 primary antibody. Plasma membrane targeting qualitatively similar to normal GLUT1 was observed for charged substitutions Lys (K), Glu (E), His (H), and, to a lesser extent, Leu (L), whereas mutant Cys (C) was associated with diminished membrane signal relative to cytoplasmic signal. Water-injected oocytes did not exhibit appreciable staining. For both A and B, molecular mass markers are labeled in kDa. The gel in A represents one of seven experiments, and B represents one of two independent experiments, all of which yielded similar results.

FIGURE 4.

Transport capacity of GLUT1 and Arg-126 mutants expressed in oocytes. Each solid bar represents 3OMG flux through oocytes injected with GLUT1 or mutant transporters after subtraction of water-injected oocyte 3OMG flux. The open bar in each panel represents the actual 3OMG flux through water-injected oocytes used for subtraction. A, influx was measured in cells exposed to 1 mm extracellular 3OMG in near zero-trans conditions. B, efflux was determined in cells injected with concentrated 3OMG to achieve ∼1 mm intracellular 3OMG. Measurements were obtained from a least three independent batches of oocytes with n ≥ 15. Error bars represent S.D.

FIGURE 5.

Concentration dependence of 3OMG influx and efflux. A, concentration dependence of 3OMG influx into oocytes expressing GLUT1 and Arg-126 mutants. B, 3OMG efflux measured from oocytes injected with 3OMG. Transport isotherms were obtained by fitting a Michaelis-Menten equation to the efflux and influx data. Measurements were obtained from a least three independent batches of oocytes with n ≥ 15. Error bars represent S.D. WT, wild type.

FIGURE 6.

Effect of pH 6.5 on 3OMG transport through GLUT1 and R126H. A and B, efflux and influx were assayed as in Fig. 4. C and D, concentration dependence of influx and efflux, respectively. Error bars represent S.D. WT, wild type; mes, methanesulfonic acid.

FIGURE 7.

Inhibition of 3OMG transport by ZnCl2 in GLUT1- (filled bars) and R126H (open bars)-expressing oocytes. Cells were preincubated in freshly dissolved ZnCl2 for 10 min prior to the assay and maintained in the same blocker concentration in the presence of 50 mm glucose during the assay. The efficacy of ZnCl2 as a blocker of GLUT1 and R126H was not significantly different (t test p > 0.1) under 10 and 100 μm ZnCl2. n ≥ 4 oocytes per assay. Error bars represent S.D.

FIGURE 8.

GLUT1 model. TM helices are represented in gray except for TM4, indicated by a yellow ribbon, and select helices, labeled and shown in color. The side chains of residues subject to missense mutation are depicted in pink and numbered. Top panel, view normal to the membrane from the extracellular aspect of the transporter. Bottom panel, view parallel to the plane of the membrane. Dotted lines represent the putative boundaries of the plasma membrane and have been arbitrarily placed. IN and OUT denote the extracellular and intracellular spaces, respectively.