The red blood cell glucose transporter presents multiple, nucleotide-sensitive sugar exit sites.
At any instant, the human erythrocyte sugar transporter presents at least one sugar export site but multiple sugar import sites. The present study asks whether the transporter also presents more than one sugar exit site. We approached this question by analysis of binding of [3H]cytochalasin B (an export conformer ligand) to the human erythrocyte sugar transporter and by analysis of cytochalasin B modulation of human red blood cell sugar uptake. Phloretin-inhibitable cytochalasin B binding to human red blood cells, to human red blood cell integral membrane proteins, and to purified human red blood cell glucose transport protein (GluT1) displays positive cooperativity at very low cytochalasin B levels. Cooperativity between sites and K(d(app)) for cytochalasin B binding are reduced in the presence of intracellular ATP. Red cell sugar uptake at subsaturating sugar levels is inhibited by high concentrations of cytochalasin B but is stimulated by lower (<20 nM) concentrations. Increasing concentrations of the e1 ligand forskolin also first stimulate then inhibit sugar uptake. Cytochalasin D (a cytochalasin B analogue that does not interact with GluT1) is without effect on sugar transport over the same concentration range. Cytochalasin B and ATP binding are synergistic. ATP (but not AMP) enhances [3H]cytochalasin B photoincorporation into GluT1 while cytochalasin B (but not cytochalasin D) enhances [gamma-32P]azidoATP photoincorporation into GluT1. We propose that the red blood cell glucose transporter is a cooperative tetramer of GluT1 proteins in which each protein presents a translocation pathway that alternates between uptake (e2) and export (e1) states but where, at any instant, two subunits must present uptake (e2) and two subunits must present exit (e1) states.
Cloherty
E K
EK
Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, Massachusetts 01655, USA.
Levine
K B
KB
Carruthers
A
A
D019325:3-O-Methylglucose / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism*; D050260:Carbohydrate Metabolism; D002241:Carbohydrates / Q000737:chemistry; D003571:Cytochalasin B / Q000378:metabolism; D004912:Erythrocytes / Q000737:chemistry / Q000378:metabolism*; D051272:Glucose Transporter Type 1; D006801:Humans; D007700:Kinetics; D008320:Maltose / Q000378:metabolism; D008956:Models, Chemical; D009004:Monosaccharide Transport Proteins / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D021381:Protein Transport
D002241:Carbohydrates; D051272:Glucose Transporter Type 1; D009004:Monosaccharide Transport Proteins; C495677:SLC2A1 protein, human; D019325:3-O-Methylglucose; D003571:Cytochalasin B; D008320:Maltose; D000255:Adenosine Triphosphate