α- and β-Monosaccharide transport in human erythrocytes

Abstract

Equilibrative sugar uptake in human erythrocytes is characterized by a rapid phase, which equilibrates 66% of the cell water, and by a slow phase, which equilibrates 33% of the cell water. This behavior has been attributed to the preferential transport of beta-sugars by erythrocytes (Leitch JM, Carruthers A. Am J Physiol Cell Physiol 292: C974-C986, 2007). The present study tests this hypothesis. The anomer theory requires that the relative compartment sizes of rapid and slow transport phases are determined by the proportions of beta- and alpha-sugar in aqueous solution. This is observed with D-glucose and 3-O-methylglucose but not with 2-deoxy-D-glucose and D-mannose. The anomer hypothesis predicts that the slow transport phase, which represents alpha-sugar transport, is eliminated when anomerization is accelerated to generate the more rapidly transported beta-sugar. Exogenous, intracellular mutarotase accelerates anomerization but has no effect on transport. The anomer hypothesis requires that transport inhibitors inhibit rapid and slow transport phases equally. This is observed with the endofacial site inhibitor cytochalasin B but not with the exofacial site inhibitors maltose or phloretin, which inhibit only the rapid phase. Direct measurement of alpha- and beta-sugar uptake demonstrates that erythrocytes transport alpha- and beta-sugars with equal avidity. These findings refute the hypothesis that erythrocytes preferentially transport beta-sugars. We demonstrate that biphasic 3-O-methylglucose equilibrium exchange kinetics refute the simple carrier hypothesis for protein-mediated sugar transport but are compatible with a fixed-site transport mechanism regulated by intracellular ATP and cell shape.

Will the original glucose transporter isoform please stand up!

“…In combination, these studies suggest either that transport is more complicated than anticipated or that previous transport measurements are technically flawed. Recent published reports confirm the former interpretation (9,74).…”

“…The second, the fast phase, describes sugar import into cytosol and accounts for two-thirds of total D-glucose uptake by red cells. The third and slowest phase describes a slowing of transport as endofacial sugar-binding sites become saturated (74). The potential significance of these results will be discussed below.…”

“…74). More recent studies demonstrate that ␣-and ␤-Dglucose are transported with equal avidity by GLUT1 (74). Answers to these questions must await crystallization of the GLUT1-D-glucose complex.…”

“…Exchange between binding sites and cavity is accelerated when binding sites are occupied by sugar via a process termed "geminate exchange". This model not only explains the stereospecificity and complexity of steadystate sugar transport (99) but also accounts for multiphasic sugar transport kinetics (74), the ability of GLUT1 to trap or occlude a sugar molecule within a central cavity (9), and cooperative, multisite ligand binding (41,71). The enhanced cooperativity of tetrameric GLUT1 may result from additional subunit interface interactions that are present in the tetramer.…”

Molecular basis of ligand recognition and transport by glucose transporters

“…6a) provide the molecular basis to address the long-standing questions on substrate recognition and transport by GLUTs. The discrimination of a-and b-D-glucose by GLUTs and whether anomerization is required has been controversial for decades [45][46][47] . The 1.5 Å structure of glucose-bound GLUT3 reveals that GLUTs can recognize both anomers, hence anomerization may not be required for D-glucose transport by GLUTs (Fig.…”