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 structure of receptors for the neurotransmitter L-glutamate. In this study, the sites of N-linked oligosaccharides on GluR6, a member of the kainate class of ionotropic glutamate receptors, were examined. Site-directed mutagenesis was utilized to alter the consensus sequence at three potential sites for N-linked glycosylation in the carboxyl-terminal half of the molecule. The presence of a carbohydrate substitution was monitored by shifts in the relative molecular weight of the mutant receptors on immunoblots. Molecular weight shifts were observed for the mutants 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. These results indicate that asparagines 515 and 720 are glycosylated and thus are likely located extracellularly. In immunocytochemical analyses of GluR6 expressed in baculovirus-infected cells, permeabilization with detergents was required for immunostaining with a carboxyl-terminal antibody, indicating that the carboxyl terminus is located intracellularly. Electrophysiological recordings of the mutant receptors expressed in human embryonic kidney cells demonstrated that the amplitudes of the kainate-activated currents mediated by the N574Q, N720Q, and the T722A mutants were not significantly different from currents mediated by wild type GluR6 receptors, while the currents mediated by the N515Q and T517A mutants were significantly depressed. Based on these findings, we propose a model for the transmembrane topology of GluR6.
Taverna
F A
FA
Medical Research Council Group on Nerve Cells and Synapses, University of Toronto, Ontario, Canada.
Wang
L Y
LY
MacDonald
J F
JF
Hampson
D R
DR
D000595:Amino Acid Sequence; D001216:Asparagine / Q000737:chemistry*; D001483:Base Sequence; D002462:Cell Membrane / Q000737:chemistry; D002478:Cells, Cultured; D005455:Fluorescent Antibody Technique; D006031:Glycosylation; D006801:Humans; D007608:Kainic Acid / Q000494:pharmacology; D008564:Membrane Potentials / Q000187:drug effects; D008956:Models, Chemical; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D009844:Oligosaccharides / Q000737:chemistry*; D011487:Protein Conformation; D017470:Receptors, Glutamate / Q000737:chemistry* / Q000187:drug effects
D009838:Oligodeoxyribonucleotides; D009844:Oligosaccharides; D017470:Receptors, Glutamate; D001216:Asparagine; D007608:Kainic Acid