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 Ͻ40 mg/dL), and decreased erythrocyte glucose uptake (56.1 Ϯ 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 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 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. (Pediatr Res 64: 538-543, 2008)

Nationwide survey of glucose transporter-1 deficiency syndrome (GLUT-1DS) in Japan

“…This study included 3 cases of GLUT-1DS with SLC2A1 missense mutations who did not exhibit the low uptake of 3-O-methyl-D-glucose by erythrocytes. Recent study demonstrated that the specific type of mutation alter GLUT confirmation and asymmetrically affects glucose flux across the cell by perturbing efflux (glucose release in CSF) than influx (uptake of glucose) [36]. A diversity of phenotypes was observed among patients with the same mutation and even within autosomal dominant family members, suggesting a complex onset pathomechanism.…”

Excellent response to acetazolamide in a case of paroxysmal dyskinesias due to GLUT1-deficiency

“…The mutation was found to be pathogenic using five in silico prediction software programs (Polyphen, Panther, SIFTV2, SNPs3D and Align GVGD). It is located close to a frequent mutation, Thr295Met, which specifically alters GLUT1 conformation and disrupts GLUT1 activity [7]. Oral acetazolamide was started at a daily dose of 500 mg, resulting in immediate and complete disappearance of the attacks.…”

A Protein Kinase C Phosphorylation Motif in GLUT1 Affects Glucose Transport and is Mutated in GLUT1 Deficiency Syndrome

“…nonsense, frame shift, splice junctions) often result in severe disease while missense mutants sometimes have more subtle clinical phenotypes (Leen et al, 2010). Even missense mutations that do not effect transporter expression or cell surface localization can cause neurological disease (Arsov et al, 2012; Wang et al, 2008). The phenotypic variability in the clinical presentation of G1D patients suggests nuances in the regulation of GLUT1-mediated glucose transport.…”