Article

Structure and Assembly Mechanism for Heteromeric Kainate Receptors

Laboratory of Cellular and Molecular Neurophysiology, Porter Neuroscience Research Center, NICHD, NIH, DHHS, Bethesda, MD 20892, USA.
Neuron (Impact Factor: 15.05). 07/2011; 71(2):319-31. DOI: 10.1016/j.neuron.2011.05.038
Source: PubMed

ABSTRACT

Native glutamate receptor ion channels are tetrameric assemblies containing two or more different subunits. NMDA receptors are obligate heteromers formed by coassembly of two or three divergent gene families. While some AMPA and kainate receptors can form functional homomeric ion channels, the KA1 and KA2 subunits are obligate heteromers which function only in combination with GluR5-7. The mechanisms controlling glutamate receptor assembly involve an initial step in which the amino terminal domains (ATD) assemble as dimers. Here, we establish by sedimentation velocity that the ATDs of GluR6 and KA2 coassemble as a heterodimer of K(d) 11 nM, 32,000-fold lower than the K(d) for homodimer formation by KA2; we solve crystal structures for the GluR6/KA2 ATD heterodimer and heterotetramer assemblies. Using these structures as a guide, we perform a mutant cycle analysis to probe the energetics of assembly and show that high-affinity ATD interactions are required for biosynthesis of functional heteromeric receptors.

Download full-text

Full-text

Available from: Mark L Mayer
  • Source
    • "To simulate a heteromer having a 2:2 stoichiometry, as is the case for GluK2/GluK5 KARs (Reiner et al. 2012), prolineswere introduced into the equivalent α-helical positions of the NaK structure on opposing subunits (i.e. A and C), in agreement with prior work on KAR heteromers (Kumar et al. 2011). Changes in cross-pore distances between the centre of mass of the α-carbons of residues 50–53 in opposing subunits are summarized in Fig. 6D. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Channel block and permeation by cytoplasmic polyamines is a common feature of many cation-selective ion-channels. Although the channel block mechanism has been studied extensively, polyamine permeation has been considered less significant as it occurs at extreme positive membrane potentials. Here, we show that kainate receptor (KAR) heteromerization and association with auxiliary proteins, Neto1 and Neto2, attenuate polyamine block by enhancing blocker permeation. Consequently, polyamine permeation and unblock occur at more negative and physiologically-relevant membrane potentials. In GluK2/GluK5 heteromers, enhanced permeation is due to a single proline residue in GluK5 that alters the dynamics of the α-helical region of the selectivity filter. The effect of auxiliary proteins is additive, therefore the structural basis of polyamine permeation and unblock is through a different mechanism. Since native receptors are thought to assemble as heteromers in complex with auxiliary proteins, our data identify an unappreciated impact of polyamine permeation in shaping the signalling properties of neuronal KARs and point to a structural mechanism that may be shared amongst other cation-selective ion-channels. This article is protected by copyright. All rights reserved.
    Full-text · Article · Dec 2015 · The Journal of Physiology
  • Source
    • "In eukaryotic pentameric LGICs (pLGICs) such as the a7 nAChR (Castillo et al., 2009), the r1 GABA C R (Wong et al., 2014) and the a1 or a1b GlyRs (Kuhse et al., 1993; Griffon et al., 1999) intersubunit interactions of the N-terminal domains were shown to be critical for assembly. Also, in heteromeric kainate receptors, high-affinity interaction domains of the KA2 and GluR6 subunits control subunit assembly (Kumar et al., 2011). The resolution of the zebrafish P2X4 receptor X-ray structure has shown the extracellular contact interfaces of adjacent subunits (Kawate et al., 2009), which has together with mutagenesis studies (Hausmann et al., 2015) shed light on the intersubunit interaction domains that control subunit assembly of P2X receptors. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The aim of the present work was to clarify whether heterotrimeric P2X2/3 receptors have a fixed subunit stoichiometry consisting of one P2X2 and two P2X3 subunits as previously suggested, or a flexible stoichiometry containing also the inverse subunit composition. For this purpose we transfected HEK293 cells with P2X2 and P2X3 encoding cDNA at the ratios of 1:2 and 4:1, and analysed the biophysical and pharmacological properties of the generated receptors by means of the whole-cell patch-clamp technique. The concentration-response curves for the selective agonist α,β-meATP did not differ from each other under the two transfection ratios. However, co-expression of an inactive P2X2 mutant and the wildtype P2X3 subunit and vice versa resulted in characteristic distortions of the α,β-meATP concentration-response relationships, depending on which subunit was expressed in excess, suggesting that HEK293 cells express mixtures of (P2X2)1/(P2X3)2 and (P2X2)2/(P2X3)1 receptors. Whereas the allosteric modulators H(+) and Zn(2+) failed to discriminate between the two possible heterotrimeric receptor variants, the α,β-meATP-induced responses were blocked more potently by the competitive antagonist A317491, when the P2X2 subunit was expressed in deficit of the P2X3 subunit. Furthermore, blue-native PAGE analysis of hP2X2 and hP2X3 subunits co-expressed in Xenopus laevis oocytes and HEK293 cells revealed that plasma membrane-bound P2X2/3 receptors appeared in two clearly distinct heterotrimeric complexes: a (P2X2-GFP)2/P2X3 complex and a P2X2-GFP/(P2X3)2 complex. These data strongly indicate that the stoichiometry of the heteromeric P2X2/3 receptor is not fixed, but determined in a permutational manner by the relative availability of P2X2 and P2X3 subunits. Copyright © 2015. Published by Elsevier Ltd.
    Full-text · Article · Jul 2015 · Neuropharmacology
  • Source
    • "The crystal structure of the AMPA GluA2 receptor (PDB ID: 3KG2) (Sobolevsky et al., 2009) was selected as the main template. Additional templates were used for the N-terminal domain (crystal structure of the GluK2/GluK5 NTD tetramer assembly, PDB ID: 3QLV) (Kumar et al., 2011) and the ligand-binding domain (crystal structure of GluK1 ligand-binding domain (S1S2) in complex with an antagonist , PDB ID: 4DLD) (Venskutonyt_ e et al., 2012). Homology modeling was carried out with Modeler v. 9.11 (Eswar et al., 2006). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Here we present the synthesis, pharmacological activity, and molecular docking of novel non-competitive antagonists of GluK2 receptor. The compounds concerned are derivatives of indole and carbazole and are the second reported series of non-competitive antagonists of the GluK2 receptor (the first one was also published by our group). The activity of the indole derivatives is in the micromolar range, as in the case of the first series of non-competitive GluK2 receptor antagonists. We have found that designed carbazole derivatives are devoid of activity. Active indole derivatives interact with the transduction domain of the GluK2 receptor, i.e., the domain which links the transmembrane region of the receptor with the agonist-binding domain. The binding pocket is situated within one receptor subunit.
    Full-text · Article · Feb 2014 · Medicinal Chemistry Research
Show more