Revised Manuscript C - 00001 - 2015 1 Caffeine inhibits glucose transport by binding at the 2 GLUT 1 nucleotide - binding site 3 4 5

23 GLUT1 is the primary glucose transport protein of the cardiovascular system and astroglia. A 24 recent study proposes that caffeine uncompetitive inhibition of GLUT1 results from interactions 25 at an exofacial GLUT1 site. Intracellular ATP is also an uncompetitive GLUT1 inhibitor and 26 shares structural similarities with caffeine suggesting that caffeine acts at the previously 27 characterized endofacial GLUT1 nucleotide-binding site. We tested this by confirming that 28 caffeine uncompetitively inhibits GLUT1-mediated 3-O-methylglucose uptake in human 29 erythrocytes (Vmax and Km for sugar uptake are reduced 4-fold; Ki(app) = 3.5 mM caffeine). ATP 30 and AMP antagonize caffeine inhibition of 3-O-methylglucose uptake in erythrocyte ghosts by 31 increasing Ki(app) for caffeine inhibition of transport from 0.9 ± 0.3 mM in the absence of 32 intracellular nucleotides to 2.6 ± 0.6 and 2.4 ± 0.5 mM in the presence of 5 mM intracellular ATP 33 or AMP respectively. Extracellular ATP has no effect on sugar uptake or its inhibition by 34 caffeine. Caffeine and ATP displace the fluorescent ATP derivative, trinitrophenyl-ATP from the 35 GLUT1 nucleotide binding site but D-glucose and the transport inhibitor cytochalasin B do not. 36 Caffeine, but not ATP, inhibits cytochalasin B binding to GLUT1. Like ATP, caffeine renders the 37 GLUT1 carboxy-terminus less accessible to peptide-directed antibodies but cytochalasin B and 38 D-glucose do not. These results suggest that the caffeine binding site bridges two non39 overlapping GLUT1 endofacial sites – the regulatory, nucleotide binding site and the 40 cytochalasin B-binding site. Caffeine binding to GLUT1 mimics the action of ATP but not 41 cytochalasin B on sugar transport. Molecular docking studies support this hypothesis. 42