Human Erythrocyte Sugar Transport is Incompatible with Available Carrier Models ² Erin K. Cloherty, Karen S. Heard, and Anthony Carruthers* Department of Biochemistry and Molecular Biology, Program in Molecular Medicine, UniVersity of Massachusetts Medical School, 373 Plantation Street, Worcester, Massachusetts 01605 ReceiVed December 28, 1995; ReVised Manuscript ReceiVed May 24, 1996 X ABSTRACT: GLUT1-mediated, passive D-glucose transport in human erythrocytes is asymmetric. V max and K m(app) for D-glucose uptake at 4 °C are 10-fold lower than V max and K m(app) for D-glucose export. Transport asymmetry is not observed for GLUT1-mediated 3-O-methylglucose transport in rat, rabbit, and avian erythrocytes and rat adipocytes where V max for sugar uptake and exit are identical. This suggests that transport asymmetry is either an intrinsic catalytic property of human GLUT1 or that factors present in human erythrocytes affect GLUT1-mediated sugar transport. In the present study we assess human erythrocyte sugar transport asymmetry by direct measurement of sugar transport rates and by analysis of the effects of intra- and extracellular sugars on cytochalasin B binding to the sugar export site. We also perform internal consistency tests to determine whether the measured, steady-state 3-O-methylglucose transport properties of human erythrocytes agree with those expected of two hypothetical models for protein-mediated sugar transport. The simple-carrier hypothesis describes a transporter that alternately exposes sugar import and sugar export pathways. The fixed-site carrier hypothesis describes a sugar transporter that simultaneously exposes sugar import and sugar export pathways. Steady-state 3-O- methylglucose transport in human erythrocytes at 4 °C is asymmetric. V max and K m(app) for sugar uptake are 10-fold lower than V max and K m(app) for sugar export. Phloretin-inhibitable cytochalasin B binding to intact red cells is unaffected by extracellular D-glucose but is competitively inhibited by intracellular D-glucose. This inhibition is reduced by 13% ( 4% when saturating extracellular D-glucose levels are also present. Assuming transport is mediated by a simple-carrier and that cytochalasin B and intracellular D-glucose binding sites are mutually exclusive, the cytochalasin B binding data are explained only if transport is almost symmetric (V max exit ) 1.4 V max entry). The cytochalasin B binding data are consistent with both symmetric and asymmetric fixed-site carriers. Analysis of 3-O-methylglucose, 2-deoxy-D- glucose, and D-glucose uptake in the presence of intracellular 3-O-methylglucose demonstrates significant divergence in experimental and theoretical transport behaviors. We conclude either that human erythrocyte sugar transport is mediated by a carrier mechanism that is fundamentally different from those considered previously or that human erythrocyte-specific factors prevent accurate determination of GLUT1-mediated sugar translocation across the cell membrane. We suggest that GLUT1-mediated sugar transport in all cells is an intrinsically symmetric process but that intracellular sugar complexation in human red cells prevents accurate determination of transport rates. The passive glucose transport system of human erythro- cytes is characterized by translocational, stereochemical, and biochemical asymmetry (Widdas, 1980). V max and K m(app) for erythrocyte D-glucose uptake into sugar-depleted cells are 5-10-fold lower than the corresponding parameters for efflux into sugar-free saline (Baker & Naftalin, 1979; Hankin et al., 1972; Lowe & Walmsley, 1986; Miller, 1968b). The transporter shows asymmetric affinities for extracellular and intracellular sugars and unique stereochemical requirements for ligand binding at endo- and exofacial binding sites (Barnett et al., 1973, 1975; Basketter & Widdas, 1978). Erythrocyte sugar transport is inhibited by intracellular trypsin but not by extracellular trypsin (Carruthers & Melchior, 1983; Coderre et al., 1995; Masaik & LeFevre, 1977). These asymmetries in translocation constants, ligand binding, and susceptibility to proteolysis are not unexpected of a transport system whose catalytic subunit is an integral membrane protein (GLUT1) 1 that lacks internal, primary structural repeats and spans the plasma membrane multiple times (Mueckler et al., 1985). What is surprising is the finding that GLUT1-mediated sugar transport in rat, rabbit, and pigeon erythrocytes and in rat adipocytes and CHO fibroblasts 2 is translocationally symmetric [V max entry ) V max exit; (Helgerson & Carruthers, 1989; Naftalin & Rist, 1991; Regen & Morgan, 1964; Simons, 1983; Taylor & Holman, 1981)]. While transport in these tissues is symmetric, the stereochemistry of substrate binding at import and export sites resembles that of the human red cell sugar transporter (Helgerson & Carruthers, 1989; Holman et al., 1981b; Holman & Rees, 1982; Simons, 1983). This asymmetry in human GLUT1-mediated erythrocyte sugar translocation could result from (1) primary structural elements specific to ² This work was supported by NIH Grant DK 36081. * Author to whom correspondence should be addressed. Tel: (508) 856-5570. FAX: (508) 856-6882 or (508) 856-4289. E-mail: anthony.carruthers@ummed.edu. X Abstract published in AdVance ACS Abstracts, July 15, 1996. 1 Abbreviations used: CHO, Chinese hamster ovary; GLUT1, human erythrocyte glucose transport protein; 2DODG, 2-deoxy-D-glucose; 3OMG, 3-O-methylglucose; CCB, cytochalasin B; EDTA, ethylene- diaminetetraacetic acid; HEPES, N-[2-hydroxyethyl]piperazine-N′-[2- ethanesulfonic acid]; Tris-HCl, tris(hydroxymethyl)aminomethane. 2 S. A. Harrison, J. M. Buxton, A. Carruthers, unpublished findings. 10411 Biochemistry 1996, 35, 10411-10421 S0006-2960(95)03077-7 CCC: $12.00 © 1996 American Chemical Society