TRANSMEMBRANE TOPOLOGY OF THE GLUTAMATE-RECEPTOR SUBUNIT GLUR6

Ionotropic glutamate receptors mediate most rapid excitatory synaptic transmission in the mammalian central nervous system. These receptors are divided into alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA), kainate, and N-methyl-D-aspartate receptors based on pharmaco logical and electrophysiological characteristics. Ionotropic receptor subunits are integral membrane proteins that have been proposed to hav e a large extracellular ligand-binding N-terminal domain, four hydroph obic transmembrane domains, and an extracellular C-terminal domain. In this study we have shown that both AMPA receptor subunits (GluR1-4) a nd kainate receptor subunits (GluR6/7) are glycosylated in adult rat b rain; however, the kainate receptor subunits are glycosylated to a gre ater extent. Examination of the sequences of AMPA and kainate receptor s revealed that kainate receptors have several additional consensus si tes for N-linked glycosylation; interestingly, one of these is located in the proposed major intracellular loop of the receptor subunits. To test the proposed transmembrane topology model for these receptors, w e have used site-specific mutagenesis of the GluR6 subunit to remove t he consensus glycosylation site located within the proposed intracellu lar loop. Mutagenesis of this site demonstrates that it is glycosylate d in transiently transfected human embryonic kidney cells, which expre ss functional kainate receptors. Since N-linked glycosylation has only been found to occur on extracellular domains of plasma membrane prote ins, these results suggest that the proposed transmembrane topology mo del for the glutamate receptor subunits is incorrect. Combining these results with other recent data, we have proposed an alternative transm embrane topology model.