Examination of sodium/glucose cotransport by using a visible glucose analogue.
The glucose derivative, 2,2,6,6-tetramethylpiperidine-1-oxylglucose (TEMPO-glucose) was synthesized and examined for its ability to substitute for glucose as a substrate for the intestinal brush border membrane Na+/glucose cotransporter. TEMPO-glucose inhibited Na(+)-dependent phlorizin binding with an apparent KI of 18 microM and Na(+)-dependent glucose uptake with an apparent KI of 70 microM. The transport competence of TEMPO-glucose was examined by using two measures of transport. The first involved comparing the reversal of trans Na+ inhibition by D-glucose and TEMPO-glucose. The second directly examined Na(+)-dependent TEMPO-glucose uptake by using TEMPO-glucose quenching of intravesicular fluorescein sulfonate fluorescence. Tryptophan fluorescence was sensitive to TEMPO-glucose in a Na(+)-dependent, glucose-inhibitable manner. The bulk of these tryptophans appeared to be located in hydrophobic environments based on Cs(+)-insensitivity. With the reconstituted cotransporter, TEMPO-glucose, and tryptophan quench reagents, the cotransporter was compared in three transport modes: zero trans uptake, zero trans uptake in the presence of a shunt of membrane potential, and substrate exchange. The results suggest that the cotransporter conformation varies depending on its mode of operation and that TEMPO-glucose may be a useful probe for localizing amino acid residues involved in glucose transport.
Peerce
B E
BE
Department of Physiology and Biophysics, University of Texas Medical Branch, Galveston 77550.
D001692:Biological Transport; D002586:Cesium / Q000737:chemistry; D003497:Cyclic N-Oxides / Q000138:chemical synthesis / Q000737:chemistry; D005453:Fluorescence; D005947:Glucose / Q000378:metabolism*; D008081:Liposomes; D012964:Sodium / Q000378:metabolism*; D013113:Spin Labels; D013379:Substrate Specificity; D014364:Tryptophan; D014634:Valinomycin / Q000737:chemistry
D003497:Cyclic N-Oxides; D008081:Liposomes; D013113:Spin Labels; D002586:Cesium; D014634:Valinomycin; D014364:Tryptophan; D012964:Sodium; D005947:Glucose; C003959:TEMPO