title: Functional studies of the T295M mutation causing Glut1 deficiency: glucose efflux preferentially affected by T295M.
abstract: Glucose transporter type 1 (Glut1) deficiency syndrome (Glut1 DS, OMIM: #606777) is characterized by infantile seizures, acquired microcephaly, developmental delay, hypoglycorrhachia (CSF glucose <40 mg/dL), and decreased erythrocyte glucose uptake (56.1 +/- 17% of control). Previously, we reported two patients with a mild Glut1 deficiency phenotype associated with a heterozygous GLUT1 T295M mutation and normal erythrocyte glucose uptake. We assessed the pathogenicity of T295M in the Xenopus laevis oocyte expression system. Under zero-trans influx conditions, the T295M Vmax (590 pmol/min/oocyte) was 79% of the WT value and the Km (14.3 mM) was increased compared with WT (9.6 mM). Under zero-trans efflux conditions, both the Vmax (1216 pmol/min/oocyte) and Km (8.8 mM) in T295M mutant Glut1 were markedly decreased in comparison to the WT values (7443 pmol/min/oocyte and 90.8 mM). Western blot analysis and confocal studies confirmed incorporation of the T295M mutant protein into the plasma membrane. The side chain of M295 is predicted to block the extracellular "gate" for glucose efflux in our Glut-1 molecular model. We conclude that the T295M mutation specifically alters Glut1 conformation and asymmetrically affects glucose flux across the cell by perturbing efflux more than influx. These findings explain the seemingly paradoxical findings of Glut1 DS with hypoglycorrhachia and "normal" erythrocyte glucose uptake.
authors.affiliation: Department of Neurology, Columbia University, New York, New York 10032, USA.
authors.forename: Dong
authors.initials: D
authors.lastname: Wang
authors.forename: Hong
authors.initials: H
authors.lastname: Yang
authors.forename: Lei
authors.initials: L
authors.lastname: Shi
authors.forename: Li
authors.initials: L
authors.lastname: Ma
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Fujii
authors.forename: Kristin
authors.initials: K
authors.lastname: Engelstad
authors.forename: Juan M
authors.initials: JM
authors.lastname: Pascual
authors.forename: Darryl C
authors.initials: DC
authors.lastname: De Vivo
mesh_terms: D019325:3-O-Methylglucose / Q000378:metabolism*; D000293:Adolescent; D000818:Animals; D002239:Carbohydrate Metabolism, Inborn Errors / Q000097:blood / Q000235:genetics / Q000378:metabolism*; D002648:Child; D004912:Erythrocytes / Q000378:metabolism; D005260:Female; D018014:Gene Transfer Techniques; D005838:Genotype; D051272:Glucose Transporter Type 1 / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008297:Male; D008958:Models, Molecular; D020125:Mutation, Missense; D010641:Phenotype; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D014982:Xenopus laevis
chemical_list: D051272:Glucose Transporter Type 1; C495677:SLC2A1 protein, human; D019325:3-O-Methylglucose