MEMBRANE TOPOLOGY OF THE C-TERMINAL HALF OF THE NEURONAL, GLIAL, AND BACTERIAL GLUTAMATE TRANSPORTER FAMILY

Secondary glutamate transporters in neuronal and glial cells in the ma mmalian central nervous system remove the excitatory neurotransmitter glutamate from the synaptic cleft and prevent the extracellular glutam ate concentration to rise above neurotoxic levels. Secondary structure prediction algorithms predict 6 trans membrane helices in the first h alf of the transporters but fail in the C-terminal half where no clear helix-loop-helix motif is resolved in the hydropathy profile of the p rimary sequences. A number of previous studies have emphasized the imp ortance of the C-terminal half of the molecules for the function. Here we determine the membrane topology of the C-terminal half of the glut amate transporters by applying the phoA gene fusion technique to the h omologous bacterial glutamate transporter of Bacillus stearothermophil us. High sequence conservation and very similar hydropathy profiles in the C-terminal half warrant a similar folding as in the glutamate tra nsporters of the mammalian central nervous system, The C-terminal half contains four putative transmembrane helices. The strong hydrophobic moment and substitution moment of the most C-terminal helix X that poi nt to opposite faces of the helix suggest that the helix faces the lip id environment with its least conserved, hydrophobic face and the inte rior of the protein with its well conserved, hydrophilic face, Residue s that were shown before to be critical for function cluster in helix X and VII.