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

Caroline L Scholefield,Palmi T Atlason,David E Jane,Elek Molnár

doi:10.1007/s11064-018-2654-0

Caroline L Scholefield, Palmi T Atlason + Show 2 more

Open Access

https://doi.org/10.1007/s11064-018-2654-0

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Journal: Neurochemical Research	Publication Date: Oct 9, 2018
Citations: 7	License type: open-access

Affiliation: University of Bristol

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.

Highlights

Kainate receptors (KARs) are members of the ionotropic glutamate receptor family [1, 2]
Subunits with impaired ligand binding domain (LBD) are able to assemble with other wild type (WT) KAR subunits with functional LBD, which enables their trafficking to the cell surface [14,15,16]
We found that mutations of conserved residues in the LBDs (GluK2-A487, GluK1-T675, GluK2-T659, GluK3T661) hindered both kainate and MG binding to all low affinity subunits of KARs

Summary

Introduction

Kainate receptors (KARs) are members of the ionotropic glutamate receptor (iGluR) family [1, 2]. Each subunit contributes to the formation of the KAR channel pore and contains a unique ligand binding domain (LBD) [5] with distinctive pharmacological properties [6]. Subunits with impaired LBD are able to assemble with other wild type (WT) KAR subunits with functional LBD, which enables their trafficking to the cell surface [14,15,16]. It has been proposed, that ligand binding in the ER is required for ER exit [17]. The link between desensitization and ER exit has been made with KAR mutants, where engineered disulfide bridges across the ligand binding domain interface blocked GluK2 desensitization and retained mutant receptors in the ER [18]

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Conclusion