Key points are not available for this paper at this time.
The Glut1 glucose transporter has been proposed to form an aqueous sugar translocation pathway through the lipid bilayer via the clustering of several 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 participation of transmembrane helix 10 in the formation of this putative aqueous tunnel was tested using cysteine-scanning mutagenesis in conjunction with the membrane-impermeant, sulfhydryl-specific reagent,p-chloromercuribenzenesulfonate (pCMBS). A series of 21 mutants was created from a fully functional, cysteine-less, parental Glut1 molecule by changing each residue within putative transmembrane segment 10 to cysteine. Each mutant was then expressed inXenopus oocytes, and its plasma membrane content, 2-deoxyglucose uptake activity, and sensitivity to pCMBS were measured. Helix 10 exhibited a highly distinctive reaction profile to scanning mutagenesis whereby cysteine substitution at residues within the cytoplasmic N-terminal half of the helix tended to increase specific transport activity, whereas substitution at residues within the exoplasmic C-terminal half of the helix tended to decrease specific transport activity. Four residues within helix 10 were clearly accessible to pCMBS as judged by inhibition or stimulation of transport activity. All four of these residues were clustered along one face of a putative α-helix. These results combined with previously published data suggest that transmembrane segment 10 of Glut1 forms part of the sugar permeation pathway. Two-dimensional models for the conformation of the 12 transmembrane helices and the exofacial glucose-binding site of Glut1 are proposed that are consistent with existing experimental data. The Glut1 glucose transporter has been proposed to form an aqueous sugar translocation pathway through the lipid bilayer via the clustering of several 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 participation of transmembrane helix 10 in the formation of this putative aqueous tunnel was tested using cysteine-scanning mutagenesis in conjunction with the membrane-impermeant, sulfhydryl-specific reagent,p-chloromercuribenzenesulfonate (pCMBS). A series of 21 mutants was created from a fully functional, cysteine-less, parental Glut1 molecule by changing each residue within putative transmembrane segment 10 to cysteine. Each mutant was then expressed inXenopus oocytes, and its plasma membrane content, 2-deoxyglucose uptake activity, and sensitivity to pCMBS were measured. Helix 10 exhibited a highly distinctive reaction profile to scanning mutagenesis whereby cysteine substitution at residues within the cytoplasmic N-terminal half of the helix tended to increase specific transport activity, whereas substitution at residues within the exoplasmic C-terminal half of the helix tended to decrease specific transport activity. Four residues within helix 10 were clearly accessible to pCMBS as judged by inhibition or stimulation of transport activity. All four of these residues were clustered along one face of a putative α-helix. These results combined with previously published data suggest that transmembrane segment 10 of Glut1 forms part of the sugar permeation pathway. Two-dimensional models for the conformation of the 12 transmembrane helices and the exofacial glucose-binding site of Glut1 are proposed that are consistent with existing experimental data. p-chloromercuribenzenesulfonate Glut1 molecule in which all six native cysteine residues were changed to either glycine or serine a Glut1 mutant constructed using the C-less parent in which a single cysteine mutation was introduced in place of one the transmembrane residues Facilitative transport of glucose into 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). Glut1, the prototype member of this family, is one of the most extensively studied of all mammalian membrane transporters (4Mueckler M. Hresko R.C. Sato M. Biochem. Soc. Trans. 1997; 25: 951-954Crossref PubMed Scopus (33) Google Scholar). Kinetic studies on human red blood cell Glut1 are mostly compatible with a simple alternating conformation mechanism for sugar transport (5Lowe A.G. Walmsley A.R. Agre P. Parker J.C. Red Blood Cell Membranes. 11. Marcel Dekker, Inc., New York1989: 597-634Google Scholar). However, clear kinetic anomalies have been observed in human erythrocytes that are either inconsistent with this hypothesis or suggest the presence of factors specific to the human erythrocyte that prevent accurate measurement of steady-state kinetic properties (6Carruthers A. Physiol. Rev. 1990; 70: 1135-1176Crossref PubMed Scopus (329) Google Scholar, 7Cloherty E.K. Heard K.S. Carruthers A. Biochemistry. 1996; 35: 10411-10421Crossref PubMed Scopus (69) Google Scholar). The human Glut1 polypeptide exhibits a molecular mass of 54,117 and contains a single N-linked oligosaccharide (8Mueckler 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 presence of 12 transmembrane segments was predicted based on analysis of the deduced amino acid sequence (8Mueckler 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), and this prediction has been experimentally verified using glycosylation-scanning mutagenesis (9Hresko 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 putative transmembrane segments possess the potential to form amphipathic α-helices, which led to the hypothesis that these amphipathic helices cluster together in the membrane to form the walls of a water-filled tunnel through which sugar traverses the fatty acyl core of the lipid bilayer (8Mueckler 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 further suggested that hydroxyl- and amide-containing amino acid side chains within these helices form the glucose binding pocket within Glut1 via the formation of hydrogen bonds with sugar hydroxyl groups, although hydrophobic interactions involving aromatic residues also appear to be important (10Barnett J.E.G. Holman G.D. Munday K.A. Biochem. J. 1973; 131: 211-221Crossref PubMed Scopus (179) Google Scholar). Several pieces of evidence are consistent with this general model for the structure of Glut1. First, glutamine 161 within helix 5 (11Mueckler M. Weng W. Kruse M. J. Biol. Chem. 1994; 269: 20533-20538Abstract Full Text PDF PubMed Google Scholar) and glutamine 282 within helix 7 (12Hashiramoto 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) both appear to participate in forming the exofacial substrate-binding site. Second, valine 165, which lies near the center of helix 5 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 (13Mueckler M. Makepeace C. J. Biol. Chem. 1997; 272: 30141-30146Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Third, tryptophan 412 within helix 11 is essential for transport activity (14Garcia J.C. Strube M. Leingang K. Keller K. Mueckler M.M. J. Biol. Chem. 1992; 267: 7770-7776Abstract Full Text PDF PubMed Google Scholar). Fourth, hydrogen exchange studies indicate that 30% of peptide hydrogen atoms are freely exposed to water in purified, reconstituted Glut1, consistent with the existence of a water-accessible permeation pathway (15Jung E.K. Chin J.J. Jung C.Y. J. Biol. Chem. 1986; 261: 9155-9160Abstract Full Text PDF PubMed Google Scholar). Fifth, cysteine-scanning mutagenesis and substituted cysteine accessibility studies implicate transmembrane segments 2 (16Olsowski A. Monden I. Krause G. Keller K. Biochemistry. 2000; 39: 2469-2474Crossref PubMed Scopus (48) Google Scholar), 5 (17Mueckler M. Makepeace C. J. Biol. Chem. 1999; 274: 10923-10926Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar), 7 (18Hruz P.W. Mueckler M.M. J. Biol. Chem. 1999; 274: 36176-36180Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar), and 11 (19) of Glut1 in the formation of an aqueous transmembrane permeation pathway. In the present study, we used cysteine-scanning mutagenesis in conjunction with a sulfhydryl-specific chemical reagent to address the role of transmembrane segment 10 in the formation the Glut1 sugar permeation pathway. Our results suggest that transmembrane segment 10 is an amphipathic α-helix with a water-accessible face that lines the exofacial portion of the sugar permeation pathway. Xenopus laevis imported African frogs were purchased from Xenopus Express (Homosassa, FL), 2-3Hdeoxyglucose and diguanosine triphosphate (mRNA cap) were purchased from Amersham Biosciences, Inc., Megascript™ RNA synthesis kit was purchased from Ambion Inc (Austin, TX), and Transformer™ site-directed mutagenesis kit was obtained fromCLONTECH (Paolo Alto, CA). Procedures for the site-directed mutagenesis and sequencing of human Glut1 cDNA and the in vitro transcription and purification of Glut1 mRNAs (20Hresko 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 (21Marshall B.A. Murata H. Hresko R.C. Mueckler M. J. Biol. Chem. 1993; 268: 26193-26199Abstract Full Text PDF PubMed Google Scholar), preparation of purified oocyte plasma membranes and indirect immunofluorescence laser confocal microscopy (14Garcia 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 (11Mueckler M. Weng W. Kruse M. J. Biol. Chem. 1994; 269: 20533-20538Abstract Full Text PDF PubMed Google Scholar), and 2-deoxyglucose uptake measurements (22Keller K. Strube M. Mueckler M. J. Biol. Chem. 1989; 264: 18884-18889Abstract Full Text PDF PubMed Google Scholar) have been described in detail previously. Stage 5Xenopus 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 pCMBS 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 4× in Barth's saline and then used for the determination of 2-3Hdeoxyglucose uptake (50 μm, 30 min at 22 °C). Plasma membranes were prepared 3 days after injection of 50 ng of mutant RNA per oocyte. Western blot analysis of each of the mutant transporters was performed on 5–10 μg of total membrane protein, and the intensity of the upper fully 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). Protein levels were normalized to the C-less Glut1 control. 2-3HDeoxyglucose uptake (pmol/oocyte/30 min) of each mutant was concomitantly determined in each set of experiments and also normalized to C-less 2-3Hdeoxyglucose uptake. Specific activity was calculated by dividing relative 2-deoxyglucose uptake by the amount of Glut1 protein (compared with the C-less transporter). Uptake data were analyzed for statistical significance using the two-tailed, unpaired Studentt test. We previously constructed a mutant human Glut1 cDNA encoding a cysteine-less (C-less) Glut1 polypeptide in which all six native cysteine residues were changed to either serine or glycine residues (13Mueckler M. Makepeace C. J. Biol. Chem. 1997; 272: 30141-30146Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). The C-less transporter expressed in Xenopus oocytes exhibits transport activity nearly indistinguishable from wild-type Glut1. We used C-less Glut1 cDNA as a template to construct single-C mutants for transmembrane segment 10. Each of the 21 residues within transmembrane segment 10 was individually changed to a cysteine residue using oligonucleotide-mediated site-directed mutagenesis, producing a series of 21 mutant Glut1 molecules, each containing only a single cysteine residue (see TableI).Table ICysteine-scanning mutagenesis of helix 10Residue No.Amino acid changeCodon change369Ile → CysATC → TGC370Val → CysGTC → TGC371Ala → CysGCC → TGC372Ile → CysATC → TGC373Phe → CysTTT → TGT374Gly → CysGGC → TGC375Phe → CysTTT → TGT376Val → CysGTG → TGC377Ala → CysGCC → TGC378Phe → CysTTC → TGC379Phe → CysTTT → TGT380Glu → CysGAA → TGC381Val → CysGTG → TGC382Gly → CysGGT → TGT383Pro → CysCCT → TGT384Gly → CysGGC → TGC385Pro → CysCCC → TGC386Ile → CysATC → TGC387Pro → CysCCA → TGC388Trp → CysTGG → TGC389Phe → CysTTC → TGCcDNA encoding cysteine-less human Glut1 was subjected to oligonucleotide-mediated, site-directed mutagenesis, a series of 21 mutant in which each of the 21 residues within transmembrane helix 10 was individually changed to cysteine. to the amino acid for human Glut1 in (8Mueckler 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). in a cDNA encoding cysteine-less human Glut1 was subjected to oligonucleotide-mediated, site-directed mutagenesis, a series of 21 mutant in which each of the 21 residues within transmembrane helix 10 was individually changed to cysteine. to the amino acid for human Glut1 in (8Mueckler 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). of the single-C mutants in the oocyte plasma membrane was by indirect immunofluorescence laser confocal microscopy and by analysis of purified oocyte plasma membranes we have observed the analysis of Glut1 half of the single-C mutants were expressed in oocyte membranes at levels to the parental C-less whereas the half were expressed at and were expressed at levels relative to the parental C-less activity the oocyte was for all 21 as determined by uptake of The relative transport normalized to the plasma membrane of each mutant (see are in with previously published (14Garcia J.C. Strube M. Leingang K. Keller K. Mueckler M.M. J. Biol. Chem. 1992; 267: 7770-7776Abstract Full Text PDF PubMed Google Scholar), amino acid substitution at tryptophan transport activity. residue appears to be in the binding of the transport (14Garcia J.C. Strube M. Leingang K. Keller K. Mueckler M.M. J. Biol. Chem. 1992; 267: 7770-7776Abstract Full Text PDF PubMed Google K. T. H. M. M. H. K. M. Yazaki Y. Y. Biochem. J. 1994; Scholar). cysteine at several residues glycine glycine and within the C-terminal half of helix 10 the transport activity, whereas cysteine substitution at several residues within the N-terminal half of Glut1 valine and in transport activity. within helix 10 appear to be to cysteine substitution relative to residues within the transmembrane helices that have been analyzed by cysteine-scanning which transmembrane residues are accessible to the aqueous and part of the sugar permeation transport activity was for each of the 21 mutants after incubation in the presence of the sulfhydryl-specific pCMBS and with the in the presence of vehicle We have previously that pCMBS the glucose permeation pathway of Glut1 and has to the exofacial site (13Mueckler M. Makepeace C. J. Biol. Chem. 1997; 272: 30141-30146Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). the transport observed in the presence of pCMBS normalized for each mutant to the activity in the absence of the reagent the the in and the in The activity of single-C mutants was after incubation with that the amino acid side chains with the pCMBS be accessible from the aqueous the activity of was by reaction of pCMBS with the cysteine side at this the transmembrane segments of Glut1 that have been analyzed by cysteine-scanning mutagenesis, helix 10 exhibits a of sensitivity residues within the exoplasmic half of the helix to be by cysteine and residues within the cytoplasmic half of the helix are by The or of this are We are of results been for membrane analysis of the results of the pCMBS inhibition experiments that the residues accessible to pCMBS from the aqueous are clustered together along one face of a putative α-helix by transmembrane segment 10 (see These results are to obtained with helices 2 (16Olsowski A. Monden I. Krause G. Keller K. Biochemistry. 2000; 39: 2469-2474Crossref PubMed Scopus (48) Google Scholar), 5 (17Mueckler M. Makepeace C. J. Biol. Chem. 1999; 274: 10923-10926Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar), and 11 Helix 7 appears to be in that contains residues to pCMBS along its that its N-terminal half is in (18Hruz P.W. Mueckler M.M. J. Biol. Chem. 1999; 274: 36176-36180Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). The residues within helix 10 that are clearly to pCMBS all in the exoplasmic half of the a to that observed with helices and all of which possess residues to the exoplasmic face that are accessible to pCMBS present in the the transmembrane helices that have been of the six residues in helix 10 that are to the exoplasmic face of the membrane appears to be accessible to as judged by a in transport activity. are at for this is that one or of these residues in with pCMBS that the reaction in a in transport is a that several of the single-C mutants activity to the cysteine and a further to pCMBS be A is that of these residues is accessible to the the face of helix 10 at its is in with transmembrane is at present to these The results of cysteine-scanning analysis and substituted cysteine accessibility studies performed on 5 of the 12 transmembrane helices of Glut1 together with published and data to models for the of the 12 transmembrane helices and for the helical cluster the exofacial glucose-binding site. A for the of the transmembrane helices in Glut1 is the of most of the the which that helices in the structure to one in the with the of helices and 2 (16Olsowski A. Monden I. Krause G. Keller K. Biochemistry. 2000; 39: 2469-2474Crossref PubMed Scopus (48) Google Scholar), 5 (17Mueckler M. Makepeace C. J. Biol. Chem. 1999; 274: 10923-10926Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar), Mueckler and C. 10 and 11 (19) of Glut1 all have a face as by substituted cysteine accessibility whereas helix 7 has residues along its (18Hruz P.W. Mueckler M.M. J. Biol. Chem. 1999; 274: 36176-36180Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). clustering of the 12 transmembrane helices that is consistent with these and experimental is in and 11 be to form an helix 7 that is consistent with the results of substituted cysteine accessibility site-directed mutagenesis and helical within helices and 11 have been in exofacial substrate binding P. Mueckler M. Biol. PubMed Scopus Google Scholar). The of helices 5 and 7 in the model is consistent with hydrogen formation hydroxyl groups of a glucose molecule in the exofacial binding pocket and residues in substrate glutamine 161 (11Mueckler M. Weng W. Kruse M. J. Biol. Chem. 1994; 269: 20533-20538Abstract Full Text PDF PubMed Google Scholar) and glutamine 282 (12Hashiramoto 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). The of helices 5 and 7 is also consistent with the that valine lies at a in transporter although this residue is in transport activity (13Mueckler M. Makepeace C. J. Biol. Chem. 1997; 272: 30141-30146Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). mutagenesis studies have that side chains valine are at 165, whereas side chains are The of valine in the model glutamine 161 and glutamine 282 is consistent with at this with hydrogen formation glucose and these The of helix 11 is consistent with a hydrophobic the of glucose and tryptophan A hydrophobic involving an aromatic of Glut1 and the of glucose was predicted by (10Barnett J.E.G. 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 tryptophan at 412 is for transport activity (14Garcia 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 indicate that an aromatic is essential at this A model for the structure of Glut1 has been proposed by F. G. J. A. P. J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). model is based on the proposed helical of the and was using an model and the model of F. G. J. A. P. J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) models place helix 7 within a water-filled by the clustering of either 7 or of the transmembrane with either or four transmembrane helices of the helical The helices are in the the of the C-terminal helices the models suggest the presence of or by the with the helix 7 a role in the The models also in model is based on experimental and is of the model of F. G. J. A. P. J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) is consistent with of the data. is to of a role for glutamine 161 in the of the model of this residue is predicted to a from the proposed translocation and from the proposed substrate-binding site. is experimental evidence that glutamine 161 is in exofacial substrate and highly amino acid at this site transport activity and the exofacial of Glut1 for a glucose (11Mueckler M. Weng W. Kruse M. J. Biol. Chem. 1994; 269: 20533-20538Abstract Full Text PDF PubMed Google Scholar). F. G. J. A. P. J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) a single-C mutant at this site as to pCMBS However, the data suggest that a single-C mutant at glutamine 161 is to pCMBS inhibition that the inhibition statistical significance of the transport activity of the single-C substitution at this site (17Mueckler M. Makepeace C. J. Biol. Chem. 1999; 274: 10923-10926Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). is consistent with the that a single-C mutant at valine 165, a residue that lies one helical turn distant from glutamine 161 and on the face of the is clearly to pCMBS inhibition (17Mueckler M. Makepeace C. J. Biol. Chem. 1999; 274: 10923-10926Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). In the valine single-C mutant is used as a control in pCMBS inhibition The results of a suggest that valine lies within the glucose permeation to the exofacial substrate-binding site (13Mueckler M. Makepeace C. J. Biol. Chem. 1997; 272: 30141-30146Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). The of valine in the model of F. G. J. A. P. J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) appears to be inconsistent with these valine is by F. G. J. A. P. J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) and is from (see F. G. J. A. P. J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). mutants that have been analyzed by site-directed mutagenesis and have been to be to pCMBS of these the model is a and be to fully for the existing data and to data as PubMed Scopus Google Scholar) a model for the glucose transporter based on using the protein, a model is to the of experimental data the structure of It also that a model for a membrane transporter based on an is of significance the and transporters and Cell Membranes. Inc., Scholar). data for member of the of membrane The model we is and a for further analysis of Glut1. The of mutagenesis and to the of transporter is by the of H.R. M. Rev. Biol. PubMed Scopus Google Scholar) on the H.R. M. Rev. Biol. PubMed Scopus Google Scholar).
Mueckler et al. (Fri,) studied this question.