A TRANSMEMBRANE MODEL FOR AN IONOTROPIC GLUTAMATE-RECEPTOR PREDICTED ON THE BASIS OF THE LOCATION OF ASPARAGINE-LINKED OLIGOSACCHARIDES

Several different models have been proposed for the transmembrane stru cture of receptors for the neurotransmitter L-glutamate. In this study , the sites of N-linked oligosaccharides on GluR6, a member of the kai nate class of ionotropic glutamate receptors, were examined. Site dire cted mutagenesis was utilized to alter the consensus sequence at three potential sites for N-linked glycosylation in the carboxyl-terminal h alf of the molecule. The presence of a carbohydrate substitution was m onitored by shifts in the relative molecular weight of the mutant rece ptors on immunoblots. Molecular weight shifts were observed for the mu tants N515Q and N720Q and for two companion mutants, T517A and T722A, which also eliminate the consensus sequence for N-linked glycosylation . No shift in molecular weight was observed in the the mutant N574Q. T hese results indicate that asparagines 515 and 720 are glycosylated an d thus are likely located extracellularly. In immunocytochemical analy ses of GluR6 expressed in baculovirus-infected cells, permeabilization with detergents was required for immunostaining with a carboxyl-termi nal antibody, indicating that the carboxyl terminus is located intrace llularly. Electrophysiological recordings of the mutant receptors expr essed in human embryonic kidney cells demonstrated that the amplitudes of the kainate-activated currents mediated by the N574Q, N720Q, and t he T722A mutants were not significantly different from currents mediat ed by wild type GluR6 receptors, while the currents mediated by the N5 15Q and T517A mutants were significantly depressed. Based on these fin dings, we propose a model for the transmembrane topology of GluR6.