The anti-oxidant, flavonoid-rich content of red wine and green tea is reported to offer protection against cancer, cardiovascular disease and diabetes. Some studies, however, show that flavonoids inhibit GLUT1-mediated, facilitative glucose transport raising the possibility that their interaction with GLUT1 and subsequent, downstream effects on carbohydrate metabolism may also impact health. The present study explores the structure/function relationships of flavonoid-GLUT1 interactions. We find that low concentrations of flavonoids act as cis-allosteric activators of sugar uptake while higher concentrations competitively inhibit sugar uptake and noncompetitively inhibit sugar exit. Studies with heterologously expressed human GLUTs 1, 3 and 4 reveal that quercetin-GLUT1 and -GLUT4 interactions are stronger than quercetin-GLUT3 interactions, that ECG is more selective for GLUT1 while EGCG is less isoform-selective. Docking studies suggest that only one flavonoid can bind to GLUT1 at any instant, but sugar transport and ligand binding studies indicate that human erythrocyte GLUT1 can bind at least two flavonoid molecules simultaneously. Quercetin and EGCG are each characterized by positive, cooperative binding whereas EGC shows negative cooperative binding. These findings support recent studies suggesting that GLUT1 forms an oligomeric complex of interacting, allosteric, alternating access transporters. We discuss how modulation of facilitative glucose transporters could contribute to the protective actions of the flavonoids against diabetes and Alzheimer's.

GLUT1-mediated, facilitated sugar transport is proposed to be an example of transport by a carrier that alternately presents exofacial (e2) and endofacial (e1) substrate binding sites, commonly referred to as the alternating access carrier model. This hypothesis is incompatible with observations of co-existent exo- and endofacial ligand binding sites, transport allostery, and e1 ligand (e.g. cytochalasin B) induced GLUT1 sugar occlusion. The fixed-site carrier model proposes co-existent, interacting e2 and e1 ligand binding sites but involves sugar translocation by geminate exchange through internal cavities. Demonstrations of membrane-resident dimeric and tetrameric GLUT1 and of e2, e1 and occluded GLUT conformations in GLUT crystals of monodisperse, detergent-solubilized proteins suggest a third model. Here, GLUT1 is an alternating access carrier but the transporter complex is a dimer of GLUT1 dimers, in which subunit interactions produce two e2 and two e1 conformers at any instant. The crystallographic structures in different conformations can be utilized to further understand the transport cycle, ligand binding behavior and complex kinetics observed in GLUT1. Specifically, the GLUT1 crystal structure and homology models based upon related major facilitator superfamily proteins were used in this study, to understand inhibitor binding, ligand binding induced GLUT1 transport allostery and the existence of helix packing/oligomerization motifs and glycine induced flexibility. These studies suggest that GLUT1 functions as an oligomeric allosteric carrier where cis-allostery is an intramolecular behavior and trans-allostery is an intermolecular behavior. Additionally, mutations of a dynamic glycine affect the turnover of the transporter while mutations to helix packing motifs affect affinity.

Recent structural studies suggest that glucose transporter 1 (GLUT1)-mediated sugar transport is mediated by an alternating access transporter that successively presents exofacial (e2) and endofacial (e1) substrate-binding sites. Transport studies, however, indicate multiple, interacting (allosteric), and co-existent, exo- and endofacial GLUT1 ligand-binding sites. The present study asks whether these contradictory conclusions result from systematic analytical error or reveal a more fundamental relationship between transporter structure and function. Here, homology modeling supported the alternating access transporter model for sugar transport by confirming at least four GLUT1 conformations, the so-called outward, outward-occluded, inward-occluded, and inward GLUT1 conformations. Results from docking analysis suggested that outward and outward-occluded conformations present multiple beta-D-glucose and maltose interaction sites, whereas inward-occluded and inward conformations present only a single beta-D-glucose interaction site. Gln-282 contributed to sugar binding in all GLUT1 conformations via hydrogen bonding. Mutating Gln-282 to alanine (Q282A) doubled the Km(app) for 2-deoxy-D-glucose uptake, eliminated cis-allostery (stimulation of sugar uptake by subsaturating extracellular maltose) but not trans-allostery (uptake stimulation by subsaturating cytochalasin B). Cis-allostery persisted, but trans-allostery was lost in an oligomerization-deficient GLUT1 variant in which we substituted membrane helix 9 with the equivalent GLUT3 sequence. Moreover, Q282A eliminated cis- allostery in the oligomerization variant. These findings reconcile contradictory conclusions from structural and transport studies by suggesting that GLUT1 is an oligomer of allosteric, alternating access transporters in which 1) cis-allostery is mediated by intra-subunit interactions and 2) trans-allostery requires inter-subunit interactions.

WZB117 (2-fluoro-6-(m-hydroxybenzoyloxy) phenyl m-hydroxybenzoate) inhibits passive sugar transport in human erythrocytes and cancer cell lines and, by limiting glycolysis, inhibits tumor growth in mice. This study explores how WZB117 inhibits the erythrocyte sugar transporter glucose transport protein 1 (GLUT1) and examines the transporter isoform specificity of inhibition. WZB117 reversibly and competitively inhibits erythrocyte 3-O-methylglucose (3MG) uptake with Ki(app) = 6 mum but is a noncompetitive inhibitor of sugar exit. Cytochalasin B (CB) is a reversible, noncompetitive inhibitor of 3MG uptake with Ki(app) = 0.3 mum but is a competitive inhibitor of sugar exit indicating that WZB117 and CB bind at exofacial and endofacial sugar binding sites, respectively. WZB117 inhibition of GLUTs expressed in HEK293 cells follows the order of potency: insulin-regulated GLUT4 - GLUT1 - neuronal GLUT3. This may explain WZB117-induced murine lipodystrophy. Molecular docking suggests the following. 1) The WZB117 binding envelopes of exofacial GLUT1 and GLUT4 conformers differ significantly. 2) GLUT1 and GLUT4 exofacial conformers present multiple, adjacent glucose binding sites that overlap with WZB117 binding envelopes. 3) The GLUT1 exofacial conformer lacks a CB binding site. 4) The inward GLUT1 conformer presents overlapping endofacial WZB117, d-glucose, and CB binding envelopes. Interrogating the GLUT1 mechanism using WZB117 reveals that subsaturating WZB117 and CB stimulate erythrocyte 3MG uptake. Extracellular WZB117 does not affect CB binding to GLUT1, but intracellular WZB117 inhibits CB binding. These findings are incompatible with the alternating conformer carrier for glucose transport but are consistent with either a multisubunit, allosteric transporter, or a transporter in which each subunit presents multiple, interacting ligand binding sites.

Glucose transporter 1 (GLUT1) is the primary glucose transport protein of the cardiovascular system and astroglia. A recent study proposes that caffeine uncompetitive inhibition of GLUT1 results from interactions at an exofacial GLUT1 site. Intracellular ATP is also an uncompetitive GLUT1 inhibitor and shares structural similarities with caffeine, suggesting that caffeine acts at the previously characterized endofacial GLUT1 nucleotide-binding site. We tested this by confirming that caffeine uncompetitively inhibits GLUT1-mediated 3-O-methylglucose uptake in human erythrocytes [Vmax and Km for transport are reduced fourfold; Ki(app) = 3.5 mM caffeine]. ATP and AMP antagonize caffeine inhibition of 3-O-methylglucose uptake in erythrocyte ghosts by increasing Ki(app) for caffeine inhibition of transport from 0.9 +/- 0.3 mM in the absence of intracellular nucleotides to 2.6 +/- 0.6 and 2.4 +/- 0.5 mM in the presence of 5 mM intracellular ATP or AMP, respectively. Extracellular ATP has no effect on sugar uptake or its inhibition by caffeine. Caffeine and ATP displace the fluorescent ATP derivative, trinitrophenyl-ATP, from the GLUT1 nucleotide-binding site, but d-glucose and the transport inhibitor cytochalasin B do not. Caffeine, but not ATP, inhibits cytochalasin B binding to GLUT1. Like ATP, caffeine renders the GLUT1 carboxy-terminus less accessible to peptide-directed antibodies, but cytochalasin B and d-glucose do not. These results suggest that the caffeine-binding site bridges two nonoverlapping GLUT1 endofacial sites-the regulatory, nucleotide-binding site and the cytochalasin B-binding site. Caffeine binding to GLUT1 mimics the action of ATP but not cytochalasin B on sugar transport. Molecular docking studies support this hypothesis.