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 cytop lasmic 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 bindi ng, and photolabeling with the exofacial label ethyl)benzoyl-1,3-bis(D -mannosyloxy)-2-propylamine (ATB-BMPA) was assayed in membranes from t ransfected cells and corrected for immunoreactivity of expressed trans porters. Exchange of Arg 92 (R92L amino acid residues are numbered acc ording to the corresponding residues in the GLUT1) or Arg 333/334 (RR3 33/4LA) reduced or suppressed transport activity with no or very littl e effect on photolabeling with ATB-BMPA and cytochalasin B binding. It is suggested that the lack of these residues selectively disturbes th e substrate-induced conformational change of the carrier during transp ort. Exchange of Glu 146 (E146D) or Arg 153 (R153L) markedly reduced t ransport activity, ATB-BMPA photolabeling, and cytochalasin B binding. Transport activity and ATB-BMPA labeling were abolished in the mutant s E329Q, E393D, and R400L, whereas binding of cytochalasin B was norma l. Thus, exchange of Glu 329, Glu 393, and Arg 400 appears to arrest t he transporter in an inward facing conformation. It is concluded that the conserved arginine and glutamate residues at the cytoplasmic surfa ce of the glucose transporter GLUT4 are essential for its appropriate conformation, and that it is the interaction of charged residues which mediates the oscillation between outward and inward facing states.