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 Kd 11 nM, 32,000-fold lower than the Kd 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.
► Control of glutamate receptor assembly by selective formation of ATD heterodimers ► Ultrahigh affinity for ATD heterodimer generates an extremely stable assembly ► Crystal structures of heteromeric kainate receptor tetramers ► Widely distributed contacts on both lobes of the ATD mediate heteromer assembly
The identification of AMPA, kainate and NMDA glutamate receptor subtypes by Watkins and colleagues underlies much of our understanding of excitatory synaptic transmission in the central nervous ...system of animals. Ongoing large scale genome sequencing projects in species for which physiological analysis of receptor function is challenging are resulting in identification of numerous eukaryotic glutamate receptor ion channels in the animal kingdom of life. On the basis of sequence similarity, these are frequently classified into the three vertebrate subtypes, initially identified using subtype selective ligands. Recent work reveals unexpected ligand binding profiles for these newly identified glutamate receptors, for example, kainate receptors on which NMDA acts as a competitive antagonist, and high affinity homomeric glycine activated glutamate receptors. Structural studies reveal that only subtle changes in the ligand binding domain, often identified only in retrospect, underlie different patterns of ligand binding, and that the biology of glutamate receptors is more complex than first anticipated.
The functional analysis of ctenophore neurotransmitter receptors, transporters, and ion channels can be greatly simplified by use of heterologous expression systems. Heterologous expression allows ...the characterization of individual membrane proteins, expressed at high levels in cells, where background activity by endogenous ion channels and transporters is with few exceptions minimal. The goal of such experiments is to gain an in-depth understanding of the behavior and regulation of individual molecular species, which is challenging in native tissue, but especially so in the case of ctenophores and other marine organisms. Coupled with transcriptome analysis, and immunohistochemical studies of receptor expression in vivo, experiments with heterologous expression systems can provide valuable insight into cellular activity, prior to more challenging functional studies on native tissues.
•Resting active and desensitized state AMPA and kainate receptor structures.•First structure of an AMPA receptor TARP complex.•NMDA receptors adopt unexpected conformations.
Chemical transmission at ...excitatory synapses in the brain is mediated by a diverse family of glutamate receptor ion channels (iGluRs), tetrameric membrane protein assemblies of molecular weight 400–600kDa. Until recently, structural information for intact iGluRs was limited to biochemically tractable homomeric receptors trapped in different conformational states. These provided key insights into the mechanisms of iGluR activation and desensitization. Structures of heteromeric AMPA and NMDA receptors, the major iGluR families in the brain, together with long awaited cryo-EM structures of an AMPA receptor TARP complex, expand this picture and reveal surprising conformational diversity, raising many fundamental and controversial questions.
This review summarizes structural studies on kainate receptors that explain unique functional properties of this receptor family. A large number of structures have been solved for ligand binding ...domain dimer assemblies, giving insight into the subtype selective pharmacology of agonists, antagonists, and allosteric modulators. Structures and biochemical studies on the amino terminal domain reveal mechanisms that play a key role in assembly of heteromeric receptors. Surprisingly, structures of full length homomeric GluK2, GluK3 and heteromeric GluK2/GluK5, receptors reveal a novel structure for the desensitized state that is strikingly different from that for AMPA receptors.
This article is part of the Neuropharmacology Special Issue on ‘Glutamate Receptors – Kainate receptors’.
•The structure of kainate receptors is reviewed and related to their unique functional properties.•Crystal structures for ligand binding domains reveal subtype specific features and sites for allosteric modulation.•Cryo-EM structures for full length receptors reveal a unique conformation for the desensitized state.
X-ray crystal structures for the soluble amino-terminal and ligand-binding domains of glutamate receptor ion channels, combined with a 3.6-Å-resolution structure of the full-length AMPA receptor ...GluA2 homotetramer, provide unique insights into the mechanisms of the assembly and function of glutamate receptor ion channels. Increasingly sophisticated biochemical, computational, and electrophysiological experiments are beginning to reveal the mechanism of action of partial agonists and suggest new models for the mechanism of action of allosteric modulators. Newly identified NMDA receptor ligands acting at novel sites offer hope for the development of subtype-selective modulators. The many unresolved issues include the role of the amino-terminal domain in AMPA receptor signaling and the mechanisms by which auxiliary proteins regulate receptor activity. The structural basis for ion permeation and ion channel block also remain areas of uncertainty, and despite substantial progress, molecular dynamics simulations have yet to reveal how glutamate binding opens the ion channel pore.
Partial agonists go molecular Mayer, Mark L.
Trends in pharmacological sciences (Regular ed.),
07/2021, Letnik:
42, Številka:
7
Journal Article
Recenzirano
Single-channel analysis previously revealed a key role for a short-lived 'flipped' state during glycine receptor activation by partial agonists. Structures solved by Yu and colleagues now reveal a ...surprising mechanism involving a partially activated agonist-bound closed state that is too long-lived to be considered the flipped state.
Ion channels activated by glutamate mediate excitatory synaptic transmission in the central nervous system. Similar to other ligand-gated ion channels, their gating cycle begins with transitions from ...a ligand-free closed state to glutamate-bound active and desensitized states. In an attempt to reveal the molecular mechanisms underlying gating, numerous structures for glutamate receptors have been solved in complexes with agonists, antagonists, allosteric modulators, and auxiliary proteins. The embarrassingly rich library of structures emerging from this work reveals very dynamic molecules with a more complex conformational spectrum than anticipated from functional studies. Unanticipated conformations solved for complexes with competitive antagonists and a lack of understanding of the structural basis for ion channel subconductance states further highlight challenges that have yet to be addressed.
Excitatory synaptic transmission in the brain is mediated by ligand-gated ion channels (iGluRs) activated by glutamate. Distinct from other neurotransmitter receptors, the extracellular domains of ...iGluRs are loosely packed assemblies with two clearly distinct layers, each of which has both local and global 2-fold axes of symmetry. By contrast, the iGluR transmembrane segments have 4-fold symmetry and share a conserved pore loop architecture found in tetrameric voltage-gated ion channels. The striking layered architecture of iGluRs revealed by the 3.6 Å resolution structure of an AMPA receptor homotetramer likely arose from gene fusion events that occurred early in evolution. Although this modular design has greatly facilitated biophysical and structural studies on individual iGluR domains, and suggested conserved mechanisms for iGluR gating, recent work is beginning to reveal unanticipated diversity in the structure, allosteric regulation, and assembly of iGluR subtypes.
Little is known about the molecular mechanisms underlying differences in the ligand binding properties of AMPA, kainate, and NMDA subtype glutamate receptors. Crystal structures of the GluR5 and ...GluR6 kainate receptor ligand binding cores in complexes with glutamate, 2S,4R-4-methylglutamate, kainate, and quisqualate have now been solved. The structures reveal that the ligand binding cavities are 40% (GluR5) and 16% (GluR6) larger than for GluR2. The binding of AMPA- and GluR5-selective agonists to GluR6 is prevented by steric occlusion, which also interferes with the high-affinity binding of 2S,4R-4-methylglutamate to AMPA receptors. Strikingly, the extent of domain closure produced by the GluR6 partial agonist kainate is only 3° less than for glutamate and 11° greater than for the GluR2 kainate complex. This, together with extensive interdomain contacts between domains 1 and 2 of GluR5 and GluR6, absent from AMPA receptors, likely contributes to the high stability of GluR5 and GluR6 kainate complexes.
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