Serine-294 and threonine-295 in the exofacial loop domain between helices 7 and 8 of glucose transporters (GLUT) are involved in the conformational alterations during the transport process.

The role of a conserved polar motif (STS) in the exofacial loop between helices 7 and 8 of GLUT4 for transporter function was investigated by site-directed mutagenesis and expression of the constructs in COS-7 cells. Reconstituted glucose-transport activity, cytochalasin B binding and photolabelling with the exofacial label 2-N4-(1-azi-2,2,2-trifluoroethyl)benzoyl-1, 3-bis-(d-mannosyloxy)-2-propylamine (ATB-BMPA) were assayed in membranes from transfected cells and corrected for immunoreactivity of expressed transporters. Replacement of Ser-294 with Ala or Thr suppressed transport activity and cytochalasin B binding.

Role of conserved arginine and glutamate residues on the cytosolic surface of glucose transporters for transporter function.

The role of conserved arginine and glutamic acid residues at the cytoplasmic surface of the GLUT4 for transporter function was investigated by site-directed mutagenesis and expression of the constructs in COS-7 cells. Reconstituted glucose transport activity, cytochalasin B binding, and photolabeling with the exofacial label 2-N4-(1-azi-2,2,2-trifluoroethyl)benzoyl-1, 3-bis(d-mannosyloxy)-2-propylamine (ATB-BMPA) was assayed in membranes from transfected cells and corrected for immunoreactivity of expressed transporters. Exchange of Arg 92 (R92L amino acid residues are numbered according to the corresponding residues in the GLUT1) or Arg 333/334 (RR333/4LA) reduced or suppressed transport activity with no or very little effect on photolabeling with ATB-BMPA and cytochalasin B binding.