Functional Studies of the T295M Mutation Causing Glut1 Deficiency: Glucose Efflux Preferentially Affected by T295M

Glucose transporter type 1 (Glut 1) deficiency syndrome (Glut1 DS, OMIM: #606777) is characterized by infantile seizures, acquired microcephaly, developmental delay, hypoglycorhachia (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 cose uptake. We assessed the pathogenicity of T295M in the Xenopus laevis oocyte expression system. Under zero-trans influx conditions. the T295M V-max (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 V-max (1216 pmol/min/oocyte) and Kin (8.8 mM) in T295M mutant Glut] 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 Glut1 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 Glut] DS With hypoglycorrhachia and normal erythrocyte glucose uptake. (Pediatr Res 64: 538-543, 2008)