Functional Characterization of a Cysteine‐Less Isoform of the Human GLUT2 Hexose Transporter

Abstract

The GLUT2 carrier is the major glucose transporter isoform expressed in hepatocytes, insulin‐secreting pancreatic beta cells, and absorptive epithelial cells of the intestinal mucosa and kidney. GLUT2 is suggested to play a critical role in maintaining glucose homeostasis because it functions as a low affinity, high‐turnover transport machinery. However, little is known regarding the mechanism of transport at the structural level. Here, we designed a cysteine‐less isoform of GLUT2 (C‐less GLUT2) to serve as platform for functional and structural studies, i.e. cysteine‐scanning mutagenesis in combination with thiol chemical modification. The kinetic properties of the C‐less GLUT2 expressed into Xenopus oocytes were like those of the native GLUT2, except that the overall activity was lower than that of the wild‐type transporter. Like the native protein, the C‐less GLUT2 transported methylglucose and deoxyglucose, and the activity was reduced in the presence of other sugars such as fructose, sorbitol, 3‐O‐methyl‐D‐glucose, galactose and ribose. Similarly, the activity of the C‐less isoform was inhibited by the classical GLUT2 inhibitors cytochalasin B, quercetin and resveratrol. This C‐less protein was refractory to MTSET, a membrane‐impermeable thiol‐alkylating reagent that obliterated the activity of native GLUT2. Our data suggest that the C‐less isoform of GLUT2 retains native‐like function and therefore is a suitable platform to perform structure‐function analysis by cysteine‐scanning mutagenesis.Support or Funding InformationSupported by grants from FONDECYT 1130386, and FONDEF D11I1131