The glucose transport activity of GLUT1 is markedly decreased by substitution of a single amino acid with a different charge at residue 415.
GLUT1 glucose transporter cDNA was modified to introduce a single amino acid substitution of aspartic acid for asparagine 415, which is conserved among all facilitative glucose transporter isoforms. Although a significant amount of the mutated transporter was expressed into plasma membranes of Chinese hamster ovary cells by transfection with expression vector, almost no increase in glucose transport activity was observed. Analysis of glucose uptake with Lineweaver-Burk plot depicts that the mutation induced a marked decrease (more than 5-fold) in turnover number and a slight increase (1.5-fold) in Km compared with the wild-type GLUT1. Results obtained with cytochalasin B and ethylidene glucose suggested that the inner but not outer glucose binding site was modulated. These results suggest that asparagine 415 is located close to the inner glucose binding site and the putative inner gate of GLUT1 glucose transporter and that an ionic charge in this domain might play an important role in the rate of conformational change between an inward-facing form and an outward-facing form of glucose transporter.
Ishihara
H
H
Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo, Japan.
Asano
T
T
Katagiri
H
H
Lin
J L
JL
Tsukuda
K
K
Shibasaki
Y
Y
Yazaki
Y
Y
Oka
Y
Y
D000818:Animals; D001216:Asparagine / Q000235:genetics; D001224:Aspartic Acid / Q000235:genetics; D001665:Binding Sites; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D006224:Cricetinae; D003412:Cricetulus; D003571:Cytochalasin B / Q000378:metabolism; D004247:DNA / Q000737:chemistry; D003847:Deoxyglucose / Q000378:metabolism; D005947:Glucose / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D007700:Kinetics; D009004:Monosaccharide Transport Proteins / Q000235:genetics*; D009154:Mutation
D009004:Monosaccharide Transport Proteins; C013984:ethylidene glucose; D001224:Aspartic Acid; D003571:Cytochalasin B; D001216:Asparagine; D004247:DNA; D003847:Deoxyglucose; D005947:Glucose