Transmembrane Exchange of Fluorosugars: Characterization of Red Cell GLUT1 Kinetics Using 19F NMR

Abstract

We have developed a new approach, to our knowledge, to quantify the equilibrium exchange kinetics of carriermediated transmembrane transport of fluorinated substrates. The method is based on adapted kinetic theory that describes the concentration dependence of the transmembrane exchange rates of two competing, simultaneously transported species. Using the new approach, we quantified the kinetics of membrane transport of both anomers of three monofluorinated glucose analogs in human erythrocytes (red blood cells) using 19 F NMR exchange spectroscopy. An inosine-based glucose-free medium was shown to promote survival and stable metabolism of red blood cells over the duration of the experiments (several hours). Earlier NMR studies only yielded the apparent rate constants and transmembrane fluxes of the anomeric species, whereas we could categorize the two anomers in terms of the catalytic activity (specificity constants) of the glucose transport protein GLUT1 toward them. Differences in the membrane permeability of the three glucose analogs were qualitatively interpreted in terms of local perturbations in the bonding of substrates to key amino acid residues in the active site of GLUT1. The methodology of this work will be applicable to studies of other carrier-mediated membrane transport processes, especially those with competition between simultaneously transported species. The GLUT1-specific results can be applied to the design of probes of glucose transport or inhibitors of glucose metabolism in cells, including those exhibiting the Warburg effect.

Structure, function and regulation of mammalian glucose transporters of the SLC2 family

“…Studies on C1-fluoro glucoses (which do not interconvert) suggest that the β anomer binds more strongly than the α isomer [14] and is transported more rapidly by GLUT1 [135]. However, additional experiments in which glucose anomer mixtures are studied [130,189] suggest that the α anomer is preferred. These experiments are complicated by rapid interconversion of α and β glucose anomers and by hetero exchanges (αβ and βα, exchanges).…”

“…The latter dominates the transport kinetics and particularly the maximum rate of transport, which is the sum of the maximum exchange rates for α + β, no matter which anomer is being followed [170]. However, the rate of transport at low substrate concentrations (or the ratio of apparent K m over apparent V max ) is clearly faster for the α anomer [189]. Furthermore, in the crystal structure of GLUT3, both α and β glucose are present but α glucose is more abundant [55].…”

Fluorinated carbohydrates as chemical probes for molecular recognition studies. Current status and perspectives

“…This is the case for the glucose (Glu1) transporter mediated transport of D-glucose over the erythrocyte membrane, which has been studied by 19 F-NMR and 19 F-2D-EXSY-NMR by various groups over the years as a model for the development of glucose analogues for imaging and tumor targeting applications. 34a, 103,104 Both techniques take advantage of the shift to higher frequencies of fluorine resonances when moving from an extracellular to an intracellular environment. 103 Where signal overlap between intra-and extracellular signals hinders peak integration, 19 F-2D-EXSY-NMR can provide the required resolving power, with the intensity of cross peaks directly proportional to the flux.…”

“…21) showed uptake rates similar to glucose and a generally enhanced transport of the a-anomer, 105 while the transport of the 4-and 6-deoxyfluoro analogues (5.2, 5.3) was only half of that of glucose. 104 In contrast, higher fluorinated compounds like the hexafluoroglucose racemic analogue 5.4 were shuttled over the erythrocyte membrane with an order of magnitude higher transport efficiency compared to glucose. 34a,101 However, a later synthesized trifluoroderivative of glucose 3.2 showed an overall decrease efflux efficiency to about 70% compared to 5.1, used as a reference.…”

Review of Mutarotase in ‘Metabolic Subculture’ and Analytical Biochemistry: Prelude to 19F NMR Studies of its Substrate Specificity and Mechanism

“…For example, using 19 F NMR magnetisation-transfer experiments, it has been shown that glucose transporter GLUT1 (in human RBCs) transports the α-anomer faster than the β-anomer, and this is likely to hold in other tissues. [8][9][10] Glucose freely enters the glomerular filtrate via GLUT1, GLUT4 and GLUT8 and passes into the convoluted tubules. From there it enters the lining cells of the tubules, via the sodium dependent glucose transporters SGLT1 and SGLT2.…”