Effect of lipid packing on the conformational states of purified GLUT-1 hexose transporter.

The purpose of this study was to determine the effect of increased lipid packing on the conformational states of the GLUT-1 hexose transporter purified in endogenous lipids. The binding of glucose results in a conformational change that can be followed by a decrease in fluorescence intensity. Lipid packing was increased by subjecting the samples to hydrostatic pressure. We have found that in the absence of ligand, the fluorescence intensity decreased approximately 20% in the 600 bar range studied. In the presence of either saturating or half-saturating amounts of D-glucose, a substantial loss in intensity (approximately 80%) was observed. Similar decreases were also seen the presence of a glucose analog, maltose, or a noncompetitive inhibitor, cytochalasin B. Changes in the accessibility of aqueous soluble quenchers (I- and acrylamide) to GLUT-1 Trp and Tyr residues suggested that ligand binding causes interfacial fluorophores to move closer to ionic groups in the lipid head group region of the membrane. This idea was substantiated by (1) increased static quenching of the GLUT-1 fluorophores in the presence of ligand, (2) increased energy transfer efficiency between GLUT-1 fluorophores and a fluorescent membrane probe located close to the head group region, and (3) reduced change in rotational motion with temperature in the presence of ligand. Since the application of pressure results in an increase in bilayer thickness, and ligand binding causes a population of fluorophores to move closer to the membrane surface, then these interfacial interactions can be more stabilized under pressure. Studies monitoring the change in quenching of membrane probes by GLUT-1 tryptophans and energy transfer of GLUT-1 tryptophans to membrane probes support this idea.

Barbiturates inhibit hexose transport in cultured mammalian cells and human erythrocytes and interact directly with purified GLUT-1.

Barbiturates reduce cerebral blood flow, metabolism, and Glc transfer across the blood-brain barrier. The effect of barbiturates on hexose transport in cultured mammalian cell lines and human erythrocytes was studied. Pentobarbital inhibits [3H]-2-dGlc uptake in 3T3-C2 murine fibroblasts by approximately 95% and approximately 50% at 10 and 0.5 mM, respectively. Uptake of [3H]-2-dGlc is linear with time in the presence or absence of pentobarbital, and the percent inhibition is constant. This suggests that hexose transport, not phosphorylation, is inhibited by barbiturates. Inhibition by pentobarbital of hexose transport in 3T3-C2 cells is rapid (< 1 min), is not readily reversible, is not altered by the presence of albumin [1% (w/v)], and is independent of temperature (4-37 degrees C) and the level of cell surface GLUT-1. The IC50's for inhibition of hexose transport in 3T3-C2 cells by pentobarbital, thiobutabarbital, and barbital are 0.8, 1.0, and 4 mM, respectively. This is consistent with both the Meyer-Overton rule and the pharmacology of barbiturates. Neither halothane (< or = 10 mM) nor ethanol [< or = 0.4% (v/v)] significantly inhibits hexose transport. Inhibition by pentobarbital (0.5 mM) of [3H]-2-dGlc uptake by 3T3-C2 cells decreases the apparent Vmax (approximately 50%) but does not alter the apparent Km (approximately 0.5 mM). Inhibition of hexose transport by barbiturates, but not ethanol [< or = 0.4% (v/v)], is also observed in human erythrocytes and four other cultured mammalian cell lines. Pentobarbital quenches (Qmax approximately 75%) the intrinsic fluorescence of purified and reconstituted GLUT-1 (Kd approximately 3 mM). Quenching is independent of Glc occupancy, is unchanged by mild proteolytic inactivation, and does not appear to directly involve perturbations of the lipid bilayer. We propose that barbiturates can interact directly with GLUT-1 and inhibit the intrinsic activity of the carrier. Glc crosses the blood-brain barrier primarily via the GLUT-1 of the endothelial cells of cerebral capillaries. Partial inhibition of this process by barbiturates may be of significance to cerebral protection.