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

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 α-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. 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 α-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. 1Baldwin S.A. Biochim. Biophys. Acta. 1993; 1154: 17-49Crossref PubMed Scopus (280) Google Scholar, 2Mueckler M. Eur. J. Biochem. 1994; 219: 713-725Crossref PubMed Scopus (958) Google Scholar, 3Pessin J.E. Bell G.I. Annu. Rev. Physiol. 1992; 54: 911-930Crossref PubMed Scopus (385) Google Scholar). The GLUT protein family belongs to the major facilitator superfamily, the largest superfamily of proteins that function as membrane transporters (4Pao S.S. Paulsen I.T. Saier Jr., M.H. Microbiol. Mol. Biol. Rev. 1998; 62: 1-34Crossref PubMed Google Scholar). GLUT1, also known as the red cell glucose transporter, is perhaps the most extensively studied of all membrane transporters (5Mueckler M. Hresko R.C. Sato M. Biochem. Soc. Trans. 1997; 25: 951-954Crossref PubMed Scopus (33) Google Scholar). Kinetic analyses of glucose transport in the human red blood cell are mostly consistent with a simple alternating conformation mechanism (6Lowe A.G. Walmsley A.R. Agre P. Parker J.C. Red Blood Cell Membranes. Vol. 11. Marcel Dekker, Inc., New York1989: 597-634Google Scholar). Although kinetic anomalies have been observed in human erythrocytes that appear to be inconsistent with this simple mechanism (7Carruthers A. Physiol. Rev. 1990; 70: 1135-1176Crossref PubMed Scopus (329) Google Scholar, 8Cloherty E.K. Heard K.S. Carruthers A. Biochemistry. 1996; 35: 10411-10421Crossref PubMed Scopus (69) Google Scholar), an alternate explanation is that the anomalies may be the result of difficulties in accurately measuring steady-state kinetic properties in the red cell. GLUT1 was predicted to possess 12 transmembrane helices based on hydrophobicity analysis of the deduced amino acid sequence (9Mueckler M. Caruso C. Baldwin S.A. Panico M. Blench I. Morris H.R. Allard W.J. Lienhard G.E. Lodish H.F. Science. 1985; 229: 941-945Crossref PubMed Scopus (1149) Google Scholar). This prediction has been confirmed by glycosylation-scanning mutagenesis (10Hresko R.C. Kruse M. Strube M. Mueckler M. J. Biol. Chem. 1994; 269: 20482-20488Abstract Full Text PDF PubMed Google Scholar). Several of the 12 proposed transmembrane segments are predicted to form amphipathic α-helices, an observation which led to the hypothesis that these helices form the walls of a water-filled chamber through which glucose permeates the lipid bilayer (9Mueckler M. Caruso C. Baldwin S.A. Panico M. Blench I. Morris H.R. Allard W.J. Lienhard G.E. Lodish H.F. Science. 1985; 229: 941-945Crossref PubMed Scopus (1149) Google Scholar). It was also proposed that the hydroxyl- and amide-containing amino acid side chains within these helices form the sugar binding site(s) of GLUT1 via hydrogen bond formation with glucose hydroxyl groups, although hydrophobic interactions between aromatic amino acid side chains and the C-6 region of glucose also appear to be important (11Barnett J.E. Holman G.D. Munday K.A. Biochem. J. 1973; 131: 211-221Crossref PubMed Scopus (179) Google Scholar). Considerable experimental support for this structural model has accumulated. Cysteine-scanning mutagenesis and substituted cysteine accessibility studies implicate transmembrane segments 2 (12Olsowski A. Monden I. Krause G. Keller K. Biochemistry. 2000; 39: 2469-2474Crossref PubMed Scopus (48) Google Scholar), 5 (13Mueckler M. Makepeace C. J. Biol. Chem. 1999; 274: 10923-10926Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar), 7 (12Olsowski A. Monden I. Krause G. Keller K. Biochemistry. 2000; 39: 2469-2474Crossref PubMed Scopus (48) Google Scholar, 14Hruz P.W. Mueckler M.M. J. Biol. Chem. 1999; 274: 36176-36180Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar), 10 (15Mueckler M. Makepeace C. J. Biol. Chem. 2002; 277: 3498-3503Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar), and 11 (16Hruz P.W. Mueckler M.M. Biochemistry. 2000; 39: 9367-9372Crossref PubMed Scopus (34) Google Scholar) of GLUT1 in the formation of a water-accessible cleft within the membrane. Glutamine 161 within helix 5 (17Mueckler M. Weng W. Kruse M. J. Biol. Chem. 1994; 269: 20533-20538Abstract Full Text PDF PubMed Google Scholar) and glutamine 282 within helix 7 (18Hashiramoto M. Kadowaki T. Clark A.E. Muraoka A. Momomura K. Sakura H. Tobe K. Akanuma Y. Yazaki Y. Holman G.D. J. Biol. Chem. 1992; 267: 17502-17507Abstract Full Text PDF PubMed Google Scholar) 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 (19Mueckler M. Makepeace C. J. Biol. Chem. 1997; 272: 30141-30146Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). An aromatic side chain at position 412 within helix 11 appears to be essential for transport activity (20Garcia J.C. Strube M. Leingang K. Keller K. Mueckler M.M. J. Biol. Chem. 1992; 267: 7770-7776Abstract Full Text PDF PubMed Google Scholar). Finally, hydrogen exchange studies demonstrate that 30% of peptide hydrogen atoms are exposed to water in purified reconstituted GLUT1, which is consistent with the presence of an aqueous cleft in the membrane (21Jung E.K. Chin J.J. Jung C.Y. J. Biol. Chem. 1986; 261: 9155-9160Abstract Full Text PDF PubMed Google Scholar). In this study cysteine-scanning mutagenesis was used in conjunction with a sulfhydryl-specific chemical reagent to examine the possible role of transmembrane segment 8 in the formation of the GLUT1 substrate translocation pathway. Our results suggest that transmembrane segment 8 is an amphipathic α-helix with a water-accessible face that lines the exofacial portion of the sugar permeation pathway. Materials—Xenopus laevis imported African frogs were purchased from Xenopus Express (Homosassa, FL). 2-[3H]Deoxyglucose and diguanosine triphosphate (mRNA cap) were purchased from Amersham Biosciences. A Megascript™ RNA synthesis kit was purchased from Ambion, Inc. (Austin, TX), and a Transformer™ site-directed mutagenesis kit was obtained from Clontech. General Procedures—Procedures for the site-directed mutagenesis and sequencing of human GLUT1 cDNA, the in vitro transcription and purification of GLUT1 mRNAs (22Hresko R.C. Murata H. Marshall B.A. Mueckler M. J. Biol. Chem. 1994; 269: 32110-32119Abstract Full Text PDF PubMed Google Scholar), isolation, microinjection, and incubation of Xenopus oocytes (23Marshall B.A. Murata H. Hresko R.C. Mueckler M. J. Biol. Chem. 1993; 268: 26193-26199Abstract Full Text PDF PubMed Google Scholar), the preparation of purified oocyte plasma membranes and indirect immunofluorescence laser confocal microscopy (20Garcia J.C. Strube M. Leingang K. Keller K. Mueckler M.M. J. Biol. Chem. 1992; 267: 7770-7776Abstract Full Text PDF PubMed Google Scholar), SDS-polyacrylamide gel electrophoresis and immunoblotting with GLUT1 C-terminal antibody (17Mueckler M. Weng W. Kruse M. J. Biol. Chem. 1994; 269: 20533-20538Abstract Full Text PDF PubMed Google Scholar), and 2-deoxyglucose uptake measurements (24Keller K. Strube M. Mueckler M. J. Biol. Chem. 1989; 264: 18884-18889Abstract Full Text PDF PubMed Google Scholar) have been described in detail previously. Treatment with pCMBS—Stage 5 Xenopus oocytes were injected with 50 ng of wild-type or mutant GLUT1 mRNA. Two days after injection, groups of ∼20 oocytes were incubated for 15 min in the presence or absence of the indicated concentrations of p-chloromercuribenzenesulfonate (pCMBS), 1The abbreviations used are: pCMBS, p-chloromercuribenzenesulfonate; C-less, a GLUT1 molecule in which all six native cysteine residues were changed to either glycine or serine; single-C, a GLUT1 mutant constructed using the C-less parent in which a single cysteine mutation was introduced in place of one the transmembrane residues. in Barth’s saline at 22 °C. The 100× concentrated reagent stock was prepared in 100% dimethyl sulfoxide, and control oocytes were treated with the appropriate concentration of vehicle alone. After a 15-min incubation period, the oocytes were washed four times in Barth’s saline and then used for the determination of 2-[3H]deoxyglucose uptake (50 μm, 30 min at 22 °C). Specific Activity Determinations—Plasma membranes were prepared 3 days following the injection of 50 ng of mutant RNA/oocyte. Western blot analysis of each of the mutant transporters was performed on ∼1 μg of total membrane protein, and the intensity of the glycosylated GLUT1 band was quantified by scanning densitometry using a Molecular Dynamics phosphorimager SI. Analysis was performed using the ImageQuant NT program (Version 4.0). 2-[3H]Deoxyglucose uptake (pmol/oocyte/30 min) of each mutant was concomitantly determined in each set of experiments. Specific activity is expressed as the 2-deoxyglucose uptake/ng of mutant GLUT1 protein expressed/μg of total oocyte membrane protein. Purified human erythrocyte membranes were loaded on the same gels as the oocyte membrane samples for use as a quantitative standard. Statistical Analysis—Uptake data were analyzed for statistical significance using the two-tailed unpaired Student’s t test. We described previously (19Mueckler M. Makepeace C. J. Biol. Chem. 1997; 272: 30141-30146Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar) the properties of a cysteine-less (C-less) human GLUT1 polypeptide in which all six native cysteine residues were changed to either serine or glycine residues. When expressed in Xenopus oocytes the C-less transporter exhibits transport activity nearly indistinguishable from wild-type GLUT1 (19Mueckler M. Makepeace C. J. Biol. Chem. 1997; 272: 30141-30146Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 25Wellner M. Monden I. Keller K. FEBS Lett. 1995; 370: 19-22Crossref PubMed Scopus (22) Google Scholar), indicating that none of the native cysteine residues plays an essential role in transport function. C-less GLUT1 cDNA was used as a template to construct single-C mutants for transmembrane segment 8. Mutant cDNAs were constructed using oligonucleotide-mediated site-directed mutagenesis in which each of the 21 residues within transmembrane segment 8 was individually changed to a cysteine residue producing 21 mutant GLUT1 molecules, each possessing only a single cysteine residue (see Table I).Table ICysteine scanning mutagenesis of helix 8 cDNA encoding cysteine-less human Glut1 was subjected to oligonucleotide-mediated, site-directed mutagenesis, creating a series of 21 mutant cDNAs in which each of the 21 residues within transmembrane helix 8 was individually changed to cysteine. Residue number refers to the amino acid numbering for human Glut1 given in Ref. 9Mueckler M. Caruso C. Baldwin S.A. Panico M. Blench I. Morris H.R. Allard W.J. Lienhard G.E. Lodish H.F. Science. 1985; 229: 941-945Crossref PubMed Scopus (1149) Google Scholar. Amino acids are designated by the single-letter code.Residue No.Amino acid changeCodon change307Val in a of the single-C mutants in the oocyte plasma membrane was confirmed by indirect immunofluorescence laser confocal microscopy and by Western blot analysis of purified oocyte membranes have observed for the analysis of GLUT1 helices (13Mueckler M. Makepeace C. J. Biol. Chem. 1999; 274: 10923-10926Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 14Hruz P.W. Mueckler M.M. J. Biol. Chem. 1999; 274: 36176-36180Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, M. Makepeace C. J. Biol. Chem. 2002; 277: 3498-3503Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, P.W. Mueckler M.M. Biochemistry. 2000; 39: 9367-9372Crossref PubMed Scopus (34) Google Scholar), the single-C mutants were expressed at concentrations in the oocyte plasma membrane the of uptake data to to the of the mutants with the C-less The of of the 21 single-C mutants was in the oocytes, the mutant was expressed at to activity the oocyte was for all mutants as determined by uptake of The uptake data are in and the transport to the plasma membrane of each mutant are in at threonine 310, and threonine transport activity to the C-less cysteine at transport activity. which transmembrane residues are accessible to the aqueous and may part of the sugar permeation transport activity was for each of the 21 mutants after incubation in the presence of the membrane-impermeant sulfhydryl-specific reagent, pCMBS We have previously (19Mueckler M. Makepeace C. J. Biol. Chem. 1997; 272: 30141-30146Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar) that pCMBS the glucose permeation pathway of GLUT1 and has to the exofacial sugar binding site. 3 the transport observed in the presence of pCMBS for each mutant to the activity in the absence of the reagent, a of of pCMBS, by pCMBS, and inhibition by The activity of four single-C mutants and was after incubation with pCMBS, indicating that the amino acid side chains with the pCMBS and be accessible to the aqueous a control for inhibition by pCMBS (13Mueckler M. Makepeace C. J. Biol. Chem. 1999; 274: 10923-10926Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, M. Makepeace C. J. Biol. Chem. 1997; 272: 30141-30146Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). analysis of the results of the pCMBS inhibition that the four residues accessible to pCMBS from the aqueous are clustered along one face of a putative α-helix by transmembrane segment 8 (see These results are to obtained with helices 2 (12Olsowski A. Monden I. Krause G. Keller K. Biochemistry. 2000; 39: 2469-2474Crossref PubMed Scopus (48) Google Scholar), 5 (13Mueckler M. Makepeace C. J. Biol. Chem. 1999; 274: 10923-10926Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar), 10 (15Mueckler M. Makepeace C. J. Biol. Chem. 2002; 277: 3498-3503Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar), and 11 (16Hruz P.W. Mueckler M.M. Biochemistry. 2000; 39: 9367-9372Crossref PubMed Scopus (34) Google Scholar). 7 appears to be in that residues to pCMBS along that is in P.W. Mueckler M.M. J. Biol. Chem. 1999; 274: 36176-36180Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). the four residues within helix 8 that are to pCMBS all within the of the a result to that observed with helices and all of which possess residues to the face that are accessible to the pCMBS in the at positions 310, and threonine in the transport activity of The side chains of threonine and are predicted to within the aqueous translocation pathway based on the orientation of the helix to pCMBS that these residues may be in hydrogen to glucose in the exofacial binding pCMBS only transport at position 310, which is inconsistent with hydrogen between glucose and the side chain of the face of the membrane of the amino acid positions that were to pCMBS is possible that helix 8 as through the membrane that an helix (see The serine side chain of this residue may be in helical via hydrogen to a residue in an helix (5Mueckler M. Hresko R.C. Sato M. Biochem. Soc. Trans. 1997; 25: 951-954Crossref PubMed Scopus (33) Google or R.C. Kruse M. Strube M. Mueckler M. J. Biol. Chem. 1994; 269: 20482-20488Abstract Full Text PDF PubMed Google Scholar). a cysteine at threonine may hydrogen between the hydroxyl of the threonine side chain and a side chain from an a cysteine at transport activity. This residue is predicted to either be in with the lipid or to helix 5 (see It is possible that the side chain at this position the of helices 5 and 8 with the transporter and that this is by activity observed with the cysteine side A major in of the of the major facilitator transporters was in the form of data at for the J. I. G. H.R. S. Science. PubMed Scopus Google Scholar) and the Y. J. M. Science. PubMed Scopus Google Scholar). were in their and in this the a with a of The data for these two members of the major facilitator superfamily are consistent with the helical model that was proposed for GLUT1 (9Mueckler M. Caruso C. Baldwin S.A. Panico M. Blench I. Morris H.R. Allard W.J. Lienhard G.E. Lodish H.F. Science. 1985; 229: 941-945Crossref PubMed Scopus (1149) Google Scholar). The also the that the 12 transmembrane transporter are from a that a on the 8 of the 12 transmembrane helices form a the substrate binding site. the structures suggest for and by simple the of helices that substrate binding are exposed to either the or An alternating conformation mechanism of this was by J. Biol. PubMed Scopus Google Scholar) nearly four based on kinetic and inhibitor J.E. Holman G.D. Munday K.A. Biochem. J. PubMed Scopus Google Scholar) and studies Lienhard G.E. Biochemistry. 1989; PubMed Scopus Google Scholar, Lienhard G.E. J. Biol. Chem. 1985; Full Text PDF PubMed Google Scholar) on the red cell glucose transporter have a All of the cysteine accessibility (12Olsowski A. Monden I. Krause G. Keller K. Biochemistry. 2000; 39: 2469-2474Crossref PubMed Scopus (48) Google Scholar, M. Makepeace C. J. Biol. Chem. 1999; 274: 10923-10926Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 14Hruz P.W. Mueckler M.M. J. Biol. Chem. 1999; 274: 36176-36180Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, M. Makepeace C. J. Biol. Chem. 2002; 277: 3498-3503Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, P.W. Mueckler M.M. Biochemistry. 2000; 39: 9367-9372Crossref PubMed Scopus (34) Google Scholar) on GLUT1 pCMBS to helical to the exofacial conformation of the transporter, and the data for the and the may be to these GLUT1 is that GLUT1 the same helical with these two bacterial transporters and that this is the transport major in the of helices in the of the membrane this use homology modeling to two-dimensional model for the exofacial substrate binding site of 2 (12Olsowski A. Monden I. Krause G. Keller K. Biochemistry. 2000; 39: 2469-2474Crossref PubMed Scopus (48) Google Scholar), 5 (13Mueckler M. Makepeace C. J. Biol. Chem. 1999; 274: 10923-10926Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar), 8 10 (15Mueckler M. Makepeace C. J. Biol. Chem. 2002; 277: 3498-3503Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar), and 11 (16Hruz P.W. Mueckler M.M. Biochemistry. 2000; 39: 9367-9372Crossref PubMed Scopus (34) Google Scholar) of GLUT1 all have a single face as by substituted cysteine accessibility helices and 7 P.W. Mueckler M.M. J. Biol. Chem. 1999; 274: 36176-36180Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar) have residues along their P.W. Mueckler M.M. J. Biol. Chem. 1999; 274: 36176-36180Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). These experimental are consistent with the helical of the J. I. G. H.R. S. Science. PubMed Scopus Google Scholar) and Y. J. M. Science. PubMed Scopus Google Scholar) in their that the transmembrane helices in the of the membrane to the The is proposed to be by helices and 5 in the of the molecule and by the helices 8, and 11 in the C-terminal A of the transmembrane helices as from the face of the membrane and in the based on homology modeling using the as a is in A putative model of the exofacial binding is in within helices and 11 have been in exofacial substrate binding (reviewed in Ref. P.W. Mueckler M.M. Mol. Biol. PubMed Scopus Google Scholar). is known to be in the exofacial binding via interactions between GLUT1 and the at positions and (11Barnett J.E. Holman G.D. Munday K.A. Biochem. J. 1973; 131: 211-221Crossref PubMed Scopus (179) Google Scholar). The orientation of helices 5 and 7 in the model is consistent with hydrogen bond formation between hydroxyl groups of a glucose molecule in the exofacial binding and two residues in substrate glutamine 161 (17Mueckler M. Weng W. Kruse M. J. Biol. Chem. 1994; 269: 20533-20538Abstract Full Text PDF PubMed Google Scholar) and glutamine 282 (18Hashiramoto M. Kadowaki T. Clark A.E. Muraoka A. Momomura K. Sakura H. Tobe K. Akanuma Y. Yazaki Y. Holman G.D. J. Biol. Chem. 1992; 267: 17502-17507Abstract Full Text PDF PubMed Google Scholar). Glutamine 282 appears to with the glucose hydroxyl S.A. Biochemistry. 1998; PubMed Scopus Google Scholar). The orientation of helix 5 is also consistent with the observation that near the of the exofacial substrate binding although this residue is in transport activity (19Mueckler M. Makepeace C. J. Biol. Chem. 1997; 272: 30141-30146Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). mutagenesis studies (19Mueckler M. Makepeace C. J. Biol. Chem. 1997; 272: 30141-30146Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar) have that side chains are at position 165, side chains are The orientation of in the model is consistent with at this position with hydrogen bond formation between glucose and glutamine The transport activity of the and mutants along with their orientation along the face of helix 8 that the side chains of these residues may be in hydrogen bond formation with glucose in the exofacial substrate binding site. The orientation of helix 11 is consistent with a hydrophobic between the C-6 region of glucose and A hydrophobic a an aromatic of GLUT1 and the C-6 region of was predicted by (11Barnett J.E. Holman G.D. Munday K.A. Biochem. J. 1973; 131: 211-221Crossref PubMed Scopus (179) Google Scholar) based on transport studies substituted glucose mutagenesis studies have that a at position 412 is for transport activity (20Garcia J.C. Strube M. Leingang K. Keller K. Mueckler M.M. J. Biol. Chem. 1992; 267: 7770-7776Abstract Full Text PDF PubMed Google Scholar), and studies that an aromatic is essential at this of the of GLUT1 in the the of the protein, a that has been for membrane protein. Although the with the bacterial transporters are the with membrane structural of and the of are In the be used to the of homology modeling based on the structures of the and