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

Abstract

The GLUT1 glucose transporter has been proposed to form an aqueous substrate translocation pathway via the clustering of several amphipathic transmembrane helices (Mueckler, M., Caruso, C., Baldwin, S. A., Panico, M., Blench, I., Morris, H. R., Allard, W. J., Lienhard, G. E., and Lodish, H. F. (1985) Science 229, 941-945). The possible role of transmembrane helix 8 in the formation of this permeation pathway was investigated using cysteine-scanning mutagenesis and the membrane-impermeant sulfhydryl-specific reagent, p-chloromercuribenzenesulfonate (pCMBS). Twenty-one GLUT1 mutants were created from a fully functional cysteine-less parental GLUT1 molecule by successively changing each residue along transmembrane segment 8 to a cysteine. The mutant proteins were then expressed in Xenopus oocytes, and their membrane concentrations, 2-deoxyglucose uptake activities, and sensitivities to pCMBS were determined. Four positions within helix 8, alanine 309, threonine 310, serine 313, and glycine 314, were accessible to pCMBS as judged by the inhibition of transport activity. All four of these residues are clustered along one face of a putative ␣-helix. These results suggest that transmembrane segment 8 of GLUT1 forms part of the sugar permeation pathway. Updated two-dimensional models for the orientation of the 12 transmembrane helices and the conformation of the exofacial glucose binding site of GLUT1 are proposed that are consistent with existing experimental data and homology modeling based on the crystal structures of two bacterial membrane transporters.Passive transport of glucose across the plasma membrane of mammalian cells is mediated by members of the GLUT (SLC2a) family of membrane glycoproteins (reviewed in Refs.

GLUT1 mutations are a cause of paroxysmal exertion-induced dyskinesias and induce hemolytic anemia by a cation leak

“…The latter 2 sites of transport might both contribute to the observed neurological symptoms, since they are both involved in the delivery of glucose to nerve cells. It is conceivable that the energy demand increases under conditions of prolonged exercise and exceeds the energy supply, which is reduced in PED patients with GLUT1 mutations, as shown by our uptake experiments and by previous functional studies of the mutated sites (30,31). This hypothesis is supported by the therapeutic success of both intravenously administered glucose during exercise and a permanent ketogenic diet, which changes the main energy source of the brain from glucose to ketone bodies, as observed in the index patient of family PED1 and his older son.…”

A GSK-3/TSC2/mTOR pathway regulates glucose uptake and GLUT1 glucose transporter expression

“…In addition, a number of investigators have shown that cellular metabolism and ATP availability play a significant role in regulating GLUT1 expression and function through both direct and indirect mechanisms. Another report suggests that direct interaction of ATP with GLUT1-specific peptide sequences can lead to conformational changes that modulate transport of glucose and inhibit degradation of GLUT1 (18,35,36). Moreover, studies in skeletal muscle have shown that expression of a constitutively active form of AMP kinase (AMPK) results in increased GLUT1 and GLUT4 protein levels (11).…”

Structure of, and functional insight into the GLUT family of membrane transporters

“…45,62,63 Cysteine scanning mutagenesis was subsequently employed to probe which residues lined this proposed central pore. [64][65][66][67][68][69][70][71][72][73][74] The resulting model indicated that TMs 2, 4, 5, 7, 8, 11, and possibly 1 and 10 formed a central aqueous transport channel for glucose, whereas TMs 3, 6, 9, and 12 formed a structural scaffold on the outside of the protein. 70 A possible substrate binding site was also proposed, involving Q161, Q282, and W412.…”