Identification of an Amino Acid Residue That Lies between the Exofacial Vestibule and Exofacial Substrate-binding Site of the Glut1 Sugar Permeation Pathway

Abstract

A valine-to-isoleucine mutation at amino acid residue 197 of Glut2 or the equivalent residue 165 of Glut1 has been shown to impair glucose transport activity. This mutation was originally discovered in the Glut2 gene of a patient with type 2 diabetes. We investigated the mechanism of the effect of this mutation on transport activity via the analysis of Glut1 mutants expressed in Xenopus oocytes combined with cysteine substitution mutagenesis and the use of cysteine-reactive chemical probes. Aliphatic side chain substitutions at position 165 that were bulkier than the native valine residue inhibited glucose transport activity, whereas substitutions of less bulky side chains had little effect on transport, suggesting a role for steric hindrance. A cysteine residue was introduced at position 165 of a functional, cysteine-less Glut1 construct, and this mutant was then tested for inhibition of transport activity by a membrane-impermeant sulfhydryl-specific reagent (p-chloromercuribenzenesulfonate). p-Chloromercuribenzenesulfonate inhibited activity of the Cys 165 mutant when it was added to the external buffer but not when it was injected directly into oocytes, indicating that this residue is accessible from the external solvent but not from the cytoplasm. Competition experiments indicated that Cys 165 lies near the exofacial substrate-binding site or directly in the sugar permeation pathway. These data provide evidence that the side chain of Val 165 , which resides in the middle of transmembrane helix 5, juts into the aqueous permeation pathway of Glut1, probably between the exofacial substrate-binding site and the outer vestibule of the pathway.

Thiol Modification of Cysteine 327 in the Eighth Transmembrane Domain of the Light Subunit xCT of the Heteromeric Cystine/Glutamate Antiporter Suggests Close Proximity to the Substrate Binding Site/Permeation Pathway

“…A similar reaction pattern has been described for the V165C mutation in GLUT1 (glucose transporter-1 (27)) and for Cys 300 in GABP (Escherichia coli ␥-aminobutyric acid permease (29)). In both cases a similar size and shape of the aromatic ring of pCMB and pCMBS and substrates of those transporters were invoked to explain the rapid access of these reagents compared with some MTS reagents (29), iodoacetate, and NEM (27,29). This can hardly apply to the xCT transporter, and as expected, millimolar concentrations of pCMB or pCMBS did not inhibit transport when assayed on the C327S mutant (data not shown).…”

“…Spontaneous reactivation of the pCMB or pCMBS inactivation was not observed but may occur at a extremely slow rate. However, these data must be interpreted with caution (27); the sulfhydryl reagents may be quenched by a large excess of thiols in the oocyte cytosol, and intracellular cysteine may be rapidly incorporated to glutathione. Moreover, as xCT mediates cystine/glutamate antiport, intracellular binding sites may be saturated by the large concentration of L-glutamate in oocytes (28), preventing access of the reagents to Cys 327 .…”

Analysis of Transmembrane Segment 8 of the GLUT1 Glucose Transporter by Cysteine-scanning Mutagenesis and Substituted Cysteine Accessibility

“…When expressed in Xenopus oocytes the C-less transporter exhibits transport activity nearly indistinguishable from wild-type GLUT1 (19,25), indicating that none of the native cysteine residues plays an essential role in transport function. 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 plasma membrane fractions for immunoblot analysis.…”

“…C-less represents the parental cysteine-less GLUT1 construct. V165C is a well characterized positive control whose activity is inhibited by pCMBS (19). Star, p Ͻ 0.01 for 2-deoxyglucose uptake in the presence versus absence of pCMBS; ND, not determined.…”

“…Glutamine 161 within helix 5 (17) and glutamine 282 within helix 7 (18) appear to participate in forming the exofacial substrate binding site. Valine 165, which is positioned one helical turn distant from glutamine 161, is accessible to aqueous sulfhydryl reagents and appears to be near the exofacial substrate binding site based on mutagenesis and inhibitor studies (19). An aromatic side chain at position 412 within helix 11 appears to be essential for transport activity (20).…”