Substrate specificity and kinetic parameters of GLUT3 in rat cerebellar granule neurons

Abstract

This study examines the apparent affinity, catalytic-centre activity (‘turnover number’) and stereospecificity of the neuronal glucose transporter GLUT3 in primary cultured cerebellar granule neurons. Using a novel variation of the 3-O-[14C]methylglucose transport assay, by measuring zero-trans kinetics at 25 °C, GLUT3 was determined to be a high-apparent-affinity, high-activity, glucose transporter with a Km of 2.87±0.23 mM (mean±S.E.M.) for 3-O-methylglucose, a Vmax of 18.7± 0.48 nmol/min per 106 cells, and a corresponding catalytic-centre activity of 853 s-1. Transport of 3-O-methylglucose was competed by glucose, mannose, 2-deoxyglucose and galactose, but not by fructose. This methodology is compared with the more common 2-[3H]deoxyglucose methodology and the [U-14C]glucose transport method. The high affinity and transport activity of the neuronal glucose transporter GLUT3 appears to be an appropriate adaptation to meet the demands of neuronal metabolism at prevailing interstitial brain glucose concentrations (1–2 mM).