Docking Studies Show That D-Glucose and Quercetin Slide through the Transporter GLUT1
2005; Elsevier BV; Volume: 281; Issue: 9 Linguagem: Inglês
10.1074/jbc.m509422200
ISSN1083-351X
AutoresPhilip Cunningham, Iram Afzal‐Ahmed, Richard J Naftalin,
Tópico(s)Pancreatic function and diabetes
ResumoOn a three-dimensional templated model of GLUT1 (Protein Data Bank code 1SUK), a molecular recognition program, AUTODOCK 3, reveals nine hexose-binding clusters spanning the entire "hydrophilic" channel. Five of these cluster sites are within 3-5 Å of 10 glucose transporter deficiency syndrome missense mutations. Another three sites are within 8 Å of two other missense mutations. d-Glucose binds to five sites in the external channel opening, with increasing affinity toward the pore center and then passes via a narrow channel into an internal vestibule containing four lower affinity sites. An external site, not adjacent to any mutation, also binding phloretin but recognizing neither d-fructose nor l-glucose, may be the main threading site for glucose uptake. Glucose exit from human erythrocytes is inhibited by quercetin (Ki = 2.4 μm) but not anionic quercetin-semiquinone. Quercetin influx is retarded by extracellular d-glucose (50 mm) but not by phloretin and accelerated by intracellular d-glucose. Quercetin docking sites are absent from the external opening but fill the entire pore center. In the inner vestibule, Glu254 and Lys256 hydrogen-bond quercetin (Ki ≈ 10 μm) but not quercetin-semiquinone. Consistent with the kinetics, this site also binds d-glucose, so quercetin displacement by glucose could accelerate quercetin influx, whereas quercetin binding here will competitively inhibit glucose efflux. β-d-Hexoses dock twice as frequently as their α-anomers to the 23 aromatic residues in the transport pathway, suggesting that endocyclic hexose hydrogens, as with maltosaccharides in maltoporins, form π-bonds with aromatic rings and slide between sites instead of being translocated via a single alternating site. On a three-dimensional templated model of GLUT1 (Protein Data Bank code 1SUK), a molecular recognition program, AUTODOCK 3, reveals nine hexose-binding clusters spanning the entire "hydrophilic" channel. Five of these cluster sites are within 3-5 Å of 10 glucose transporter deficiency syndrome missense mutations. Another three sites are within 8 Å of two other missense mutations. d-Glucose binds to five sites in the external channel opening, with increasing affinity toward the pore center and then passes via a narrow channel into an internal vestibule containing four lower affinity sites. An external site, not adjacent to any mutation, also binding phloretin but recognizing neither d-fructose nor l-glucose, may be the main threading site for glucose uptake. Glucose exit from human erythrocytes is inhibited by quercetin (Ki = 2.4 μm) but not anionic quercetin-semiquinone. Quercetin influx is retarded by extracellular d-glucose (50 mm) but not by phloretin and accelerated by intracellular d-glucose. Quercetin docking sites are absent from the external opening but fill the entire pore center. In the inner vestibule, Glu254 and Lys256 hydrogen-bond quercetin (Ki ≈ 10 μm) but not quercetin-semiquinone. Consistent with the kinetics, this site also binds d-glucose, so quercetin displacement by glucose could accelerate quercetin influx, whereas quercetin binding here will competitively inhibit glucose efflux. β-d-Hexoses dock twice as frequently as their α-anomers to the 23 aromatic residues in the transport pathway, suggesting that endocyclic hexose hydrogens, as with maltosaccharides in maltoporins, form π-bonds with aromatic rings and slide between sites instead of being translocated via a single alternating site. The glucose uniporter GLUT1 (SLC2A1), a member of the major facilitator superfamily of solute transporters, has to date not been crystallized, but its three-dimensional structure has been modeled by templating it to that of Lac Y permease and glycerol 3-phosphate antiporter (GlpT) from Escherichia coli (1Abramson J. Smirnova I. Kasho V. Verner G. Iwata S. Kaback H.R. Science. 2003; 301: 610-615Crossref PubMed Scopus (1218) Google Scholar, 2Hirai T. Heymann J.A. Shi D. Sarker R. Maloney P.C. Subramaniam S. Nat. Struct. Biol. 2002; 9: 597-600PubMed Google Scholar, 3Salas-Burgos A. Iserovich P. Zuniga F. Vera J.C. Fischbarg J. Biophys. J. 2004; 87: 2990-2999Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). The 12 transmembrane α-helical domains of the monomeric GLUT protein are arranged around a central water-filled pore lined predominantly with uncharged hydrophilic and hydrophobic amino acids. The 15-Å-long, 8-Å-wide channel narrows near its midpoint (1Abramson J. Smirnova I. Kasho V. Verner G. Iwata S. Kaback H.R. Science. 2003; 301: 610-615Crossref PubMed Scopus (1218) Google Scholar, 2Hirai T. Heymann J.A. Shi D. Sarker R. Maloney P.C. Subramaniam S. Nat. Struct. Biol. 2002; 9: 597-600PubMed Google Scholar). Molecular dynamic simulations show that glucose binds close to this position within the pore, as expected of lactose binding to Lac Y permease (2Hirai T. Heymann J.A. Shi D. Sarker R. Maloney P.C. Subramaniam S. Nat. Struct. Biol. 2002; 9: 597-600PubMed Google Scholar). Glucose docks additionally in a cavity at the external entrance of the pore (3Salas-Burgos A. Iserovich P. Zuniga F. Vera J.C. Fischbarg J. Biophys. J. 2004; 87: 2990-2999Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). GLUTs transport other substrates besides hexoses (e.g. dehydroascorbate (4Vera J.C Rivas C.I. Fischbarg J. Golde D.W. Nature. 2000; 364: 79-82Crossref Scopus (446) Google Scholar, 5Rumsey S.C. Daruwala R. Al-Hasani H. Zarnowski M.J. Simpson I.A. Levine M. J. Biol. Chem. 2000; 275: 28246-28253Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar) via GLUT1, -3, and -4 and glucosamine (6Uldry M. Ibbertson M. Hosokawa M. Thorens B. FEBS Lett. 2002; 524: 199-203Crossref PubMed Scopus (220) Google Scholar) via GLUT2). The flavonone, quercetin, is transported via GLUT4. Quercetin influx into GLUT4 is inhibited by high glucose or cytochalasin B concentrations (7Strobel P. Allard C. Perez-Acle T. Calderon R. Aldunate R. Leighton F. Biochem. J. 2005; 386: 471-478Crossref PubMed Scopus (193) Google Scholar). Conversely, quercetin inhibits glucose and ascorbate transport via GLUT1, -2, -3, and -4 (8Park J.B. Levine M. J. Nutr. 2000; 130: 1297-1302Crossref PubMed Scopus (63) Google Scholar, 9Song J. Kwon O. Chen S. Daruwala R. Eck P. Park J.B. Levine M. J. Biol. Chem. 2002; 277: 15252-15260Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar, 10Naftalin R.J. Afzal I. Cunningham P. Halai M. Ross C Salleh N. Milligan S.R. Br. J. Pharmacol. 2003; 140: 487-499Crossref PubMed Scopus (56) Google Scholar). This present study demonstrates that quercetin is also transported via GLUT1, and its uptake is accelerated by exchange with intracellular glucose. Our goal here is to deduce the transport mechanism of glucose and quercetin by correlating their transport with their binding properties determined using the molecular recognition program AUTODOCK 3 (11Morris G.M. Goodsell D.S. Halliday R.S. Huey R. Hart W.E. Belew R.K. Olson A.J. J. Comput. Chem. 1998; 19: 1639-1662Crossref Scopus (9045) Google Scholar). The conventional view of passive facilitated sugar transport is that sugar binds at a single centrally located site in GLUT that isomerizes between inward and outward facing conformations. The altered direction of ligand dissociation from the site mediates transport (12Barrett M.P. Walmsley A.R. Gould G.W. Curr. Opin. Cell Biol. 1999; 11: 496-502Crossref PubMed Scopus (65) Google Scholar, 13Carruthers A. Physiol. Rev. 1990; 70: 1135-1176Crossref PubMed Scopus (324) Google Scholar, 14Abramson J. Smirnova I. Kasho V. Verner G. Iwata S. Kaback H.R. FEBS Lett. 2003; 555: 96-101Crossref PubMed Scopus (91) Google Scholar, 15Lowe A.G. Walmsley A.R. Biochim. Biophys. Acta. 1986; 857: 146-154Crossref PubMed Scopus (144) Google Scholar). To explain accelerated exchange, where the maximal rate of glucose exchange with an intracellular ligand is faster than net flux, the glucose-loaded carrier must isomerize faster than the unliganded carrier. Glucose transport asymmetry is evident as a lower maximal rate and Km for net uptake than for exit. Asymmetric transport by the mobile carrier requires that isomerization rates of the unloaded carrier are also asymmetric (16Baker G.F. Widdas W.F. J. Physiol. 1973; 231: 143-165Crossref PubMed Scopus (79) Google Scholar, 17Geck P. Biochim. Biophys. Acta. 1971; 241: 462-472Crossref PubMed Scopus (60) Google Scholar). These assumptions require that all ligands should have the same maximal transport rate, since this is determined by the slow return rate of the unloaded carrier to complete the net transport cycle (18LeFevre P.G. Am. J. Physiol. 1962; 203: 286-290Crossref PubMed Scopus (23) Google Scholar). However, differences have been observed between maximal rates of net influx of different sugars and their temperature coefficients or activation energies (19Naftalin R.J. Rist R.J. Biochim. Biophys. Acta. 1994; 1191: 65-78Crossref PubMed Scopus (15) Google Scholar, 20Cloherty E.K. Heard K.S. Carruthers A. Biochemistry. 1996; 35: 10411-10421Crossref PubMed Scopus (69) Google Scholar, 21Naftalin R.J. Biochim. Biophys. Acta. 1997; 1328: 13-29Crossref PubMed Scopus (8) Google Scholar). Another explanation for sugar transport asymmetry is that glucose accumulates in an endofacial compartment or vestibule. This promotes futile transport cycling, or recycling, that both lowers the maximal net influx rate and reduces the apparent Km for net glucose uptake (22Baker G.F. Naftalin R.J. Biochim. Biophys. Acta. 1979; 550: 474-484Crossref PubMed Scopus (68) Google Scholar, 23Levine K.B Cloherty E.K Fidyk N.J. Carruthers A. Biochemistry. 1998; 37: 12221-12232Crossref PubMed Scopus (51) Google Scholar, 24Heard K.S. Fidyk N. Carruthers A. Biochemistry. 2000; 39: 3005-3014Crossref PubMed Scopus (56) Google Scholar, 25Blodgett D.M. Carruthers A. Biochemistry. 2005; 44: 2650-2660Crossref PubMed Scopus (21) Google Scholar). A high glucose concentration binding in the vestibule widens the vestibular exit to the cytosol, whereas ATP binding restricts it, thereby altering glucose mobility between the vestibule and cytoplasmic compartment. Kinetic evidence in support of an ATP and glucose-modulated endofacial vestibular compartment (25Blodgett D.M. Carruthers A. Biochemistry. 2005; 44: 2650-2660Crossref PubMed Scopus (21) Google Scholar) is corroborated by the three-dimensional structure of GLUT1 (3Salas-Burgos A. Iserovich P. Zuniga F. Vera J.C. Fischbarg J. Biophys. J. 2004; 87: 2990-2999Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). An alternative model for glucose transport is that hexoses diffuse between recognition sites in the transport pathway independently of protein conformational changes. This multisite model accounts for saturation and inhibition kinetics and relaxes the requirement for uniform maximal rates of hexose transport (26Naftalin R.J. Afzal I. Browning J.A. Wilkins R.J. Ellory J.C. J. Membr. Biol. 2002; 186: 113-129Crossref PubMed Scopus (7) Google Scholar). Attention has been drawn to the structural similarity between GLUT1 and maltoporins (3Salas-Burgos A. Iserovich P. Zuniga F. Vera J.C. Fischbarg J. Biophys. J. 2004; 87: 2990-2999Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). Large numbers of aromatic amino acids line the "greasy pore" of maltoporin and the central pore of the GLUT1. Maltosaccharides are guided through maltoporin by slippage between hydrophobic axial hydrogen atoms projecting from the planar surface of the glycosidic ring and π-electrons of aromatic amino acid side chains in one of the maltoporin strands lining the pore. Hydrogen bonds between OH or endocyclic oxygen groups on both sides of the pyranose ring and hydrophilic porin tracks constitute the other two support rails required to guide the maltosaccharide train through the porin (27Dutzler R. Schirmer T. Karplus M. Fischer S. Structure (Camb.). 2002; 10: 1273-1284Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 28Van Gelder P. Dumas F. Bartoldus I Saint N. Prilipov A. Winterhalter M. Wang Y. Philippsen A. Rosenbusch J.P. Schirmer T. J. Bacteriol. 2002; 184: 2994-2999Crossref PubMed Scopus (35) Google Scholar). Docking and sequence comparisons here reveal that glucose transport across GLUT1 resembles sugar transport across maltoporin in that multiple docking sites provide the nodal points in a network spanning the pore length. The homologies between maltoporin and GLUT1 sequences (29Pearson W.R. Lipman D.J. Proc. Natl. Acad. Sci. U. S. A. 1988; 852: 444-2448Google Scholar) may exemplify the phenotypic convergence required of any structure that facilitates slippage of pyranose ligands through a narrow orifice rather than an ontological relationship. GLUT1 deficiency syndrome (GLUT1-DS) 3The abbreviations used are: GLUT1-DS, GLUT1 deficiency syndrome; TM, transmembrane. 3The abbreviations used are: GLUT1-DS, GLUT1 deficiency syndrome; TM, transmembrane. is caused by an inadequate energy supply to brain with failure of growth and development of the central nervous system resulting from diminished glucose transport across the blood brain barrier via defective endothelial GLUT1. The children affected tend to develop microcephaly, epileptic seizures, and ataxia. The major biochemical signs of GLUT1-DS are low cerebrospinal fluid glucose and lactate concentrations typically accompanied by low rates of glucose transport in the subjects' erythrocytes as a result of defective GLUT1 present in these cells (30Wang D. Pascual J.M. Yang H. Engelstad K. Jhung S. Sun R.P. De Vivo D.C. Ann. Neurol. 2005; 57: 111-118Crossref PubMed Scopus (260) Google Scholar, 31Klepper J. Salas-Burgos A. Gertsen E. Fischbarg J. Biochemistry. 2005; 44: 12621-12626Crossref PubMed Scopus (5) Google Scholar). Early introduction of a ketogenic diet may ameliorate this condition by supplying the brain with an alternative energy source. Sixteen missense mutations resulting in amino acid substitutions at 12 sites have been observed in 6 of the 10 exons of GLUT1 (30Wang D. Pascual J.M. Yang H. Engelstad K. Jhung S. Sun R.P. De Vivo D.C. Ann. Neurol. 2005; 57: 111-118Crossref PubMed Scopus (260) Google Scholar, 31Klepper J. Salas-Burgos A. Gertsen E. Fischbarg J. Biochemistry. 2005; 44: 12621-12626Crossref PubMed Scopus (5) Google Scholar). When mapped on the three-dimensional GLUT1 structure, these mutations are close to all but one of the hexose docking sites revealed by AUTODOCK 3. In this paper, it is shown that one of the glucose binding sites in the inner vestibule of GLUT1 that also binds uncharged quercetin is negatively charged. Docking studies reveal that this site is likely to involve Glu254 and Lys256 that hydrogen-bond to quercetin and glucose. The site is situated appropriately to allow for both retardation of net glucose and quercetin transport, so that displacement of quercetin from it by intracellular glucose explains the observed acceleration of quercetin influx. This new model of transport via GLUT1 built on previous transport, structural, and modeling studies (1Abramson J. Smirnova I. Kasho V. Verner G. Iwata S. Kaback H.R. Science. 2003; 301: 610-615Crossref PubMed Scopus (1218) Google Scholar, 2Hirai T. Heymann J.A. Shi D. Sarker R. Maloney P.C. Subramaniam S. Nat. Struct. Biol. 2002; 9: 597-600PubMed Google Scholar, 3Salas-Burgos A. Iserovich P. Zuniga F. Vera J.C. Fischbarg J. Biophys. J. 2004; 87: 2990-2999Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 7Strobel P. Allard C. Perez-Acle T. Calderon R. Aldunate R. Leighton F. Biochem. J. 2005; 386: 471-478Crossref PubMed Scopus (193) Google Scholar, 10Naftalin R.J. Afzal I. Cunningham P. Halai M. Ross C Salleh N. Milligan S.R. Br. J. Pharmacol. 2003; 140: 487-499Crossref PubMed Scopus (56) Google Scholar, 20Cloherty E.K. Heard K.S. Carruthers A. Biochemistry. 1996; 35: 10411-10421Crossref PubMed Scopus (69) Google Scholar, 22Baker G.F. Naftalin R.J. Biochim. Biophys. Acta. 1979; 550: 474-484Crossref PubMed Scopus (68) Google Scholar, 23Levine K.B Cloherty E.K Fidyk N.J. Carruthers A. Biochemistry. 1998; 37: 12221-12232Crossref PubMed Scopus (51) Google Scholar, 24Heard K.S. Fidyk N. Carruthers A. Biochemistry. 2000; 39: 3005-3014Crossref PubMed Scopus (56) Google Scholar, 25Blodgett D.M. Carruthers A. Biochemistry. 2005; 44: 2650-2660Crossref PubMed Scopus (21) Google Scholar, 26Naftalin R.J. Afzal I. Browning J.A. Wilkins R.J. Ellory J.C. J. Membr. Biol. 2002; 186: 113-129Crossref PubMed Scopus (7) Google Scholar, 27Dutzler R. Schirmer T. Karplus M. Fischer S. Structure (Camb.). 2002; 10: 1273-1284Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 28Van Gelder P. Dumas F. Bartoldus I Saint N. Prilipov A. Winterhalter M. Wang Y. Philippsen A. Rosenbusch J.P. Schirmer T. J. Bacteriol. 2002; 184: 2994-2999Crossref PubMed Scopus (35) Google Scholar) delineates how glucose and quercetin may slide via a pore structure and is clearly differentiated from the conventional alternating carrier description of glucose transport. It offers an explanation for the widely dispersed mutation sites affecting GLUT1-DS (30Wang D. Pascual J.M. Yang H. Engelstad K. Jhung S. Sun R.P. De Vivo D.C. Ann. Neurol. 2005; 57: 111-118Crossref PubMed Scopus (260) Google Scholar, 31Klepper J. Salas-Burgos A. Gertsen E. Fischbarg J. Biochemistry. 2005; 44: 12621-12626Crossref PubMed Scopus (5) Google Scholar). Fresh human erythrocytes obtained by venepuncture from a healthy donor after informed consent and approval by the King's College London Research Ethics Committee, were washed three times in isotonic saline by repeated centrifugation and resuspension. Materials—Phosphate-buffered saline tablets, d-glucose, quercetin, phloretin, ascorbate, ferricyanide, and HgCl2 were all purchased from Sigma. Stopping solution consisted of phosphate-buffered saline with 10 μm HgCl2 at 4 °C (19Naftalin R.J. Rist R.J. Biochim. Biophys. Acta. 1994; 1191: 65-78Crossref PubMed Scopus (15) Google Scholar). Measuring Glucose Efflux by Photometry—Glucose efflux from human erythrocytes preloaded with 100 mm glucose at 24 °C was measured photometrically, as previously described (10Naftalin R.J. Afzal I. Cunningham P. Halai M. Ross C Salleh N. Milligan S.R. Br. J. Pharmacol. 2003; 140: 487-499Crossref PubMed Scopus (56) Google Scholar, 21Naftalin R.J. Biochim. Biophys. Acta. 1997; 1328: 13-29Crossref PubMed Scopus (8) Google Scholar, 26Naftalin R.J. Afzal I. Browning J.A. Wilkins R.J. Ellory J.C. J. Membr. Biol. 2002; 186: 113-129Crossref PubMed Scopus (7) Google Scholar). Efflux was measured after 7.5 μl of 90% cell suspension was added to 3 ml of glucose-free saline in a fluorescence cuvette 2-3 times/s over a 5-min interval. Quercetin Uptake—Net uptake of quercetin (final concentration 25 μm) into glucose-loaded, or glucose-free red cells in 10% hematocrit at 4 °C was terminated by the addition of 200 μl of the cell suspension to ice-cold tubes containing 200 μl of stopping solution. The cell suspensions were twice washed by centrifugation at 4 °C in fresh stopping solution and then resuspended in 400 μl of phosphate-buffered saline and left for 24 h at 4 °C to allow intracellular quercetin to leak into the supernatant. The cells were then recentrifuged, and the supernatants were transferred to a 96-well plate. Quercetin in the supernatants was determined by the absorption at 380 nm with a microplate spectrophotometer. Docking Studies—Docking with the various ligands was carried out using AUTODOCK 3 (11Morris G.M. Goodsell D.S. Halliday R.S. Huey R. Hart W.E. Belew R.K. Olson A.J. J. Comput. Chem. 1998; 19: 1639-1662Crossref Scopus (9045) Google Scholar). This estimates the energy of ligand binding to the site and its Ki on a rigid three-dimensional construction of the protein and allows for rotations of the ligand bonds where applicable. Data on the position, frequency, and affinity of the ligand docking across the entire GLUT1 surface are obtained and used to map the amino acid residues within3Åofthe ligand binding sites and later the coincidences and differences between different ligands. The three-dimensional data files were collected from the following sources: GLUT1 (1SUK) and maltoporin (1AF6) from the Protein Data Bank (via the Macromolecular Structure Database on the World Wide Web at www.ebi.ac.uk/msd/). Protein Data Bank files of sugars were from "SWEET" on the World Wide Web at www.dkfz-heidelberg.de/spec (supported by the German Research Council) and from Daresbury Chemical Data base Service via "CrystalWeb" at www.cds.dl.ac.uk/cweb. Other Web tools used were the Dundee PRODRG2 Server at www.davapc1.bioch.dundee.ac.uk/prodrg. Molecular displays were created by Swiss-PdbViewer (available at ca.expasy.org/spdbv/) and RasMol (available at openrasmol.org/). The default settings of AUTODOCK were normally used, with the exception of the run and the population size. Between 80 and 100 runs were usually performed. Docking involved a grid of 101 points in three dimensions with a spacing of 0.375 Å centered on the GLUT1 molecule. When restricting the docking to sites with sparse ligand binding, the cube side was reduced to 51 units with the same spacing and centered at the coordinates of the region under consideration. Sequence Homologies between Maltoporin Ligand Binding Strands and the Central Pore Region of GLUT1—Homologies between glucose binding sequences in GLUT1 and the ligand binding strands of maltoporin were obtained as follows: the 492 amino acids of GLUT1 (1SUK) were split into 20-mers, with an overlap of 10 amino acids. The program, FASTA (29Pearson W.R. Lipman D.J. Proc. Natl. Acad. Sci. U. S. A. 1988; 852: 444-2448Google Scholar) was used to identify and evaluate the partial matches between each of the 20-mers and sequences in one of the maltoporin subunits (1AF6). The searches were then restricted to those matching the pore region GLUT1, as predicted from the templated model of GLUT1 (3Salas-Burgos A. Iserovich P. Zuniga F. Vera J.C. Fischbarg J. Biophys. J. 2004; 87: 2990-2999Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar), and the docking data for d-glucose α- and β-anomers were obtained. Coincidence between d-Glucose Docking Sites and GLUT1-DS Mutation Sites—The sites of GLUT1-DS mutations (30Wang D. Pascual J.M. Yang H. Engelstad K. Jhung S. Sun R.P. De Vivo D.C. Ann. Neurol. 2005; 57: 111-118Crossref PubMed Scopus (260) Google Scholar, 31Klepper J. Salas-Burgos A. Gertsen E. Fischbarg J. Biochemistry. 2005; 44: 12621-12626Crossref PubMed Scopus (5) Google Scholar) were mapped on the three-dimensional structure of GLUT1, and their coincidence was estimated by layering this map onto others with the α- and β-d-glucose docking sites obtained with AUTODOCK. The distances of the mutations from the hexose clusters were obtained using the measuring tools available with Swiss-PdbViewer "Deepview." Quercetin Inhibition of Glucose Transport—Inhibition of glucose transport by quercetin and other flavonols decreases when the pH is raised above 7.5 (32Salter D.W. Custead-Jones S. Cook J.S. J. Membr. Biol. 1978; 40: 67-76Crossref PubMed Scopus (27) Google Scholar). Quercetin, like genistein and estradiol, inhibits the maximal glucose exit rates without significant effect on glucose affinity at the external site, indicating that it acts on glucose transport at the endofacial surface (10Naftalin R.J. Afzal I. Cunningham P. Halai M. Ross C Salleh N. Milligan S.R. Br. J. Pharmacol. 2003; 140: 487-499Crossref PubMed Scopus (56) Google Scholar, 33Afzal I. Cunningham P. Naftalin R.J. Biochem. J. 2002; 365: 707-719Crossref PubMed Scopus (45) Google Scholar). Quercetin takes part in redox reactions with both ferricyanide and ascorbate to form the anion quercetin semiquinone (34Metodiewa D. Jaiswal A.K. Cenas N. Dickancaite E. Segura-Aguilar J. Free Radic. Biol. Med. 1999; 26: 107-116Crossref PubMed Scopus (446) Google Scholar, 35Galati G. Sabzevari O. Wilson J.X. O'Brien P.J. Toxicology. 2002; 177: 91-104Crossref PubMed Scopus (464) Google Scholar). The addition of ferricyanide or ascorbate to quercetin solutions prevents quercetin from inhibiting glucose transport. Neither ferricyanide, nor ascorbate alone, has any effect on glucose transport. This indicates that quercetin-semiquinone does not inhibit glucose transport (Fig. 1, A and B) and corroborates the view that negatively charged quercetin does not inhibit glucose transport (36Martin H.J. Kornmann F. Fuhrmann G.F. Chem. Biol. Interact. 2003; 146: 225-235Crossref PubMed Scopus (63) Google Scholar). Quercetin Uptake into Red Cells at 4 °C—High glucose concentrations or cytochalasin B inhibits quercetin uptake into adipocytes via GLUT4 (7Strobel P. Allard C. Perez-Acle T. Calderon R. Aldunate R. Leighton F. Biochem. J. 2005; 386: 471-478Crossref PubMed Scopus (193) Google Scholar). External glucose (50 mm) inhibits the quercetin (25 μm) uptake rate by 40% in human erythrocytes at 4 °C (Fig. 1C). However, when 50 mm glucose is preloaded in the internal solution and the external solution is maintained nominally glucose-free, the quercetin uptake rate is accelerated. This acceleration of quercetin uptake in the infinite-trans mode confirms that glucose and quercetin share a common transport path. Although phloretin (100 μm) inhibits glucose influx by more than 90%, it has no effect on quercetin influx with or without intracellular glucose present (not shown). Docking Studies on GLUT1: Hexoses—d-Glucose affinity for the GLUT1 transport system is ∼5 times higher than either d-mannose or d-galactose; d-fructose has a very low affinity and l-glucose has negligible affinity for GLUT1 (37Burant C.F. Bell G.I. Biochemistry. 1992; 31: 10414-10420Crossref PubMed Scopus (157) Google Scholar, 38Cohen N.R. Knecht D.A. Lodish H.F. Biochem. J. 1996; 315: 971-975Crossref PubMed Scopus (18) Google Scholar). The locations of amino acids with similar selectivity to the above were obtained using docking studies with d-glucose, d-galactose, d-mannose, and d-fructose ligands on the GLUT1 three-dimensional template. In aqueous solution, hexoses coexist as α- and β-anomers in a 34:66% ratio, respectively (39Corchado J.C. Sanchez M.L. Aguilar M.A. J. Am. Chem. Soc. 2004; 126: 7311-7319Crossref PubMed Scopus (75) Google Scholar). β-d-Glucose is transported faster than α-d-glucose by human erythrocytes (40Miwa I. Fujii H. Okuda J. Biochem. Int. 1988; 16: 111-117PubMed Google Scholar). Consequently, docking of α- and β-anomers of d-glucose, d-galactose, d-mannose, and l-glucose was studied. Docking reveals 10 clusters of sites for α- and β-d-glucose anomers on GLUT1. The clusters were identified, initially by visual inspection of the docking output of AUTODOCK 3 (Fig. 2A) and then confirmed with K-means cluster analysis (Table 1). Nine clusters are in the central hydrophilic pore region and another in a side pocket accessible from the endofacial vestibule. The α- and β-d-glucose affinities increase with depth in the external vestibule (clusters 1-5; Ki range 0.34-0.11 mm; Table 1). The increase in affinity with depth favors sugar movement from the external solution to the bottom of an energy well at cluster 5 (yellow) at the narrowest part of the channel before emergence into the internal vestibule. A similar affinity gradient to that found here with d-glucose has been observed in the maltoporin pore with maltosaccharides (27Dutzler R. Schirmer T. Karplus M. Fischer S. Structure (Camb.). 2002; 10: 1273-1284Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). The clusters in the internal vestibule (clusters 6-9) all have lower affinities (see Table 1). The cluster (green cluster 10) in the side pocket has the highest affinity, Ki = 0.04 mm. All but the red (cluster 1) and green (cluster 10) clusters are either coincident with or close to missense mutations found in GLUT1-DS (30Wang D. Pascual J.M. Yang H. Engelstad K. Jhung S. Sun R.P. De Vivo D.C. Ann. Neurol. 2005; 57: 111-118Crossref PubMed Scopus (260) Google Scholar, 31Klepper J. Salas-Burgos A. Gertsen E. Fischbarg J. Biochemistry. 2005; 44: 12621-12626Crossref PubMed Scopus (5) Google Scholar) (see "Discussion" and Table 4).TABLE 1Cluster positions of d-glucose on GLUT1 established by K means cluster analysisCluster established by K means cluster analysisRed 1Orange 2Dark blue 3Purple 4Yellow 5Gray 6Cyan 7Light blue 8Dark red 9Green 10x49.941.736.934.934.528.023.014.217.036.2y31.424.116.726.519.615.021.17.010.75.8z−24.9−26.7−24.42−39.0−32.0−32.9−45.352.0−42.8r.m.s. distance (Å)16.09.410.710.412.010.314.717.622.622.4Scale71435268109Ki (mM)0.250.340.200.110.120.550.500.440.420.04S.E. (mM)0.040.050.010.010.020.20.130.150.070.00Sugar preferencesd-Glu >>> 0-Fru-0-L-GluGlu = Mann > Gal 0 FruGlu = Mann > GalGlu = Gal > MannGlu > Gal = MannGlu > Gal = MannGlu > Gal = Mann 0 FruGlu = Gal > MannGlu = Mann > GalHigh affinity for all sugars Open table in a new tab TABLE 4Mutation analysis The missense mutations of human glucose transport deficiency syndrome GLUT1-DS in this table were obtained from Refs. 30Wang D. Pascual J.M. Yang H. Engelstad K. Jhung S. Sun R.P. De Vivo D.C. Ann. Neurol. 2005; 57: 111-118Crossref PubMed Scopus (260) Google Scholar and 31Klepper J. Salas-Burgos A. Gertsen E. Fischbarg J. Biochemistry. 2005; 44: 12621-12626Crossref PubMed Scopus (5) Google Scholar.Distance to nearest cluster (Å)Red 1Orange 2Dark blue 3Purple 4Yellow 5Gray 6Cyan 7Light blue 8Dark red 9Green 100M295T 4.9R126C 4.8T3101 3.5R153C 7.9G130S 3.1E146K 9.6K25V 2.9E247D 8.7R333W 11.5S66F 3.9R126C 5G75W (31Klepper J. Salas-Burgos A. Gertsen E. Fischbarg J. Biochemistry. 2005; 44: 12621-12626Crossref PubMed Scopus (5) Google Scholar) 4.8G91D 11.1K256V 7.7N341 2.4N341 1.9 Open table in a new tab Assumi
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