TY - JOUR
T1 - Transmembrane Exchange of Fluorosugars
T2 - Characterization of Red Cell GLUT1 Kinetics Using 19F NMR
AU - Shishmarev, Dmitry
AU - Fontenelle, Clément Q.
AU - Kuprov, Ilya
AU - Linclau, Bruno
AU - Kuchel, Philip W.
N1 - Publisher Copyright:
© 2018 Biophysical Society
PY - 2018/11/20
Y1 - 2018/11/20
N2 - We have developed a new approach, to our knowledge, to quantify the equilibrium exchange kinetics of carrier-mediated 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 19F 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.
AB - We have developed a new approach, to our knowledge, to quantify the equilibrium exchange kinetics of carrier-mediated 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 19F 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.
UR - http://www.scopus.com/inward/record.url?scp=85055083898&partnerID=8YFLogxK
U2 - 10.1016/j.bpj.2018.09.030
DO - 10.1016/j.bpj.2018.09.030
M3 - Article
SN - 0006-3495
VL - 115
SP - 1906
EP - 1919
JO - Biophysical Journal
JF - Biophysical Journal
IS - 10
ER -