Assembly and Trafficking of Homomeric and Heteromeric Kainate Receptors with Impaired Ligand Binding Sites.
Kainate receptors (KARs) are a subfamily of ionotropic glutamate receptors (iGluRs) mediating excitatory synaptic transmission. Cell surface expressed KARs modulate the excitability of neuronal networks. The transfer of iGluRs from the endoplasmic reticulum (ER) to the cell surface requires occupation of the agonist binding sites. Here we used molecular modelling to produce a range of ligand binding domain (LBD) point mutants of GluK1-3 KAR subunits with and without altered agonist efficacy to further investigate the role of glutamate binding in surface trafficking and activation of homomeric and heteromeric KARs using endoglycosidase digestion, cell surface biotinylation and imaging of changes in intracellular Ca2+ concentration [Ca2+]i. Mutations of conserved amino acid residues in the LBD that disrupt agonist binding to GluK1-3 (GluK1-T675V, GluK2-A487L, GluK2-T659V and GluK3-T661V) reduced both the total expression levels and cell surface delivery of all of these mutant subunits compared to the corresponding wild type in transiently transfected human embryonic kidney 293 (HEK293) cells. In contrast, the exchange of non-conserved residues in the LBD that convert antagonist selectivity of GluK1-3 (GluK1-T503A, GluK2-A487T, GluK3-T489A, GluK1-N705S/S706N, GluK2-S689N/N690S, GluK3-N691S) did not alter the biosynthesis and trafficking of subunit proteins. Co-assembly of mutant GluK2 with an impaired LBD and wild type GluK5 subunits enables the cell surface expression of both subunits. However, [Ca2+]i imaging indicates that the occupancy of both GluK2 and GluK5 LBDs is required for the full activation of GluK2/GluK5 heteromeric KAR channels.
['Scholefield|Caroline L|CL|', 'Atlason|Palmi T|PT|', 'Jane|David E|DE|', 'Molnár|Elek|E|http://orcid.org/0000-0002-3525-4671']
D001665:Binding Sites D002462:Cell Membrane D018698:Glutamic Acid D057809:HEK293 Cells D006801:Humans D008024:Ligands D009154:Mutation D055503:Protein Multimerization D021122:Protein Subunits D021381:Protein Transport D018092:Receptors, Kainic Acid
Trafficking Kainate receptors Ligand binding Glutamate Ca2+ imaging Site-directed mutagenesis Subunit assembly