Glucose and Water Transport by Mutants at the 66 Residue of the Glut-1 Glucose Transporter

Abstract

Abstract: : Purpose: We have reported the changes in glucose transport and water permeability resulting from the Glut 1 pathogenic mutation T310I. We report here on the functional results of four mutations at position 66, one of them also pathological. Methods: Xenopus laevis oocytes were injected with 50 nl of water or 50 nl (50 ng) of cRNA encoding Glut1 WT or S66-mutants (F, A, T, Y). Three days after injection we determined the rates of transport of glucose (³H-labeled deoxy-glucose, DOG) and water. We evaluated osmotic permeability (Pf) by challenging oocytes with hyposmotic medium (178 to 15 mOsm). Results: Mutations S66F and S66Y decreased DOG transport 15-17 fold, and increased Pf 4-fold. We also found that the increase in Pf in the S66F mutant can be competitively inhibited with [DOG] = 20mM. These results are similar to those obtained previously with the pathological T310I mutation. According to our 3d model of GLUT1, polar amino acids S66 and T310 are located at the entrances of the two channels respectively (main and auxiliary) and their side chains face the main and auxiliary water accessible channels, respectively. We also found that substitution of polar Ser66 with a nonpolar Ala of similar size had little effect on glucose transport but resulted in an increase in Pf. Conclusions: 1) The fact that S66F mutant GLUT-1 showed significantly decreased glucose transport and increased water transport, supports the hypothesis that glucose traverses GLUT1 through water- accessible pores. 2) Water and glucose compete for this pore. 3) Our proposed 3d model for GLUT1 can explain the complex effects observed.

Keywords: proteins encoded by disease genes • protein structure/function • pump/barrier function