Assembly and Trafficking of Homomeric and Heteromeric Kainate Receptors with Impaired Ligand Binding Sites

Abstract

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.

Damaging coding variants within kainate receptor channel genes are enriched in individuals with schizophrenia, autism and intellectual disabilities

“…The GluK2 LBD K525E mutation led to creation of a hydrogen bond but no predicted observable thermodynamic effect was predicted (∆∆G = 0.06), shown in Supplementary Figure S4. However, a decrease in predicted positive electrostatic potential was observed over the ligand binding domain area of the GluK2 K525E variant (Supplementary Figure S5) and which could influence cell surface expression 47 . Taken together, in silico protein modeling analysis suggests that these three predicted damaging mutations could affect protein conformation, structural relationships or electrostatic potential.…”

The Role of Glutamate Receptors in Epilepsy

“…Other protein reacting substances such as PKC (protein kinase C)-mediated phosphorylation and SUMOylation (small ubiquitin-related modifier) have been reported to have impacts on surface expression and endocytosis [ 54 ]. Moreover, molecular modeling and calcium imaging studies showed that the occupancy of both GluK2 and GluK5 ligand binding domains is required for the full activation of GluK2/GluK5 heteromeric KAR channels [ 55 ]. Of note, the non-canonical signaling of KARs which activates phospholipase C and PKC via a metabotropic G-protein dependent pathway, regulating neuronal excitability by inhibiting the slow afterhyperpolarization, neurotransmitter release, and glutamate receptor trafficking could potentially contribute to increased neuronal excitability [ 56 ].…”

Subunit-selective iGluR antagonists can potentiate heteromeric receptor responses by blocking desensitization

“…It has been shown that coexpression of GluK2 and GluK5 subunits yields functional heteromers with defined 2:2 stoichiometry next to GluK2 homotetramers (58). GluK5 homotetramers remain nonfunctional and do not traffic to the membrane (56)(57)(58)(59).…”