1.A.10 The Glutamate-gated Ion Channel (GIC) Family of Neurotransmitter Receptors
Members of the GIC family are homo or heterotetrameric complexes in which each of the 4 subunits is of 800-1000 amino acyl residues in length (Mayer, 2006). They have a modular architecture with four domains: the intracellular C-terminal domain (CTD) that is involved in synaptic targeting, the transmembrane domain (TMD) that forms the ion channel, the membrane-proximal ligand-binding domain (LBD) that binds agonists such as L-glutamate, and the distal N-terminal domain (NTD). The extracellular portion, comprised of the LBD and NTD, is loosely arranged, mediating complex allosteric regulation (Krieger et al. 2015). The structures of these receptor-channels have been reviewed with emphasis on their function and pharmacology (Regan et al. 2015). A 'hydrophobic box' in both AMPA and NMDA receptors plays a role in channel desensitization (Alsaloum et al. 2016). Activation and desensitization of ionotropic glutamate receptors by selectively triggering pre-existing motions have been proposed (Krieger et al. 2019). At least some members of this family (e.g., 1.A.10.1.10) and at least some of the metabolomic G-protein receptors (e.g., TC# 9.A.14.15.3) share an ANF receptor family, ligand binding region/domain (M. Saier, unpublished observation).
Structures of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) and N-methyl-D-aspartate (NMDA) receptors permit a comparative analysis of whole-receptor dynamics (Dutta et al. 2015). AMPA-Rs purified from the brain are tightly associated with members of the stargazin/TARP (transmembrane AMPA receptor regulatory protein) family (Nakagawa et al. 2006). In the hetero-tetrameric AMPA-R without associated stargazin/TARP proteins, the density representing the transmembrane region is substantially smaller (Nakagawa et al. 2006). Functional tetra-heteromeric NMDA receptor contains two obligatory GluN1 subunits and two identical or different non-GluN1 subunits that evolved from six different genes including four GluN2 (A-D) and two GluN3 (A-B) subunits. Since NMDA receptors confer varied physiological properties and spatiotemporal distributions in the brain, pharmacological agents that target NMDA receptors with GluN2 subunits have potential for therapeutic applications. The GluN1/2A ligand binding domain (LBD) interface interactions play a key role in determining channel function, and subtle changes in LBD interactions can be readily translated to the downstream extracellular vestibule of channel pore to adopt a conformation that may affect memantine, Zn2+ and Mg2+ binding.
Despite substantial differences in the packing of their two-domain extracellular regions, the two iGluRs share similar dynamics, elucidated by elastic network models. Motions accessible to either structure enable conformational interconversion, such as compression of the AMPA receptor toward the more tightly packed NMDA receptor conformation, which has been linked to allosteric regulation. Pivoting motions coupled to concerted rotations of the transmembrane ion channel are prominent between dimers of distal N-terminal domains in the loosely packed AMPA receptor (Dutta et al. 2015). The molecular mechanisms behind the transition of the NMDA receptor from the state where the TMSs and the ion channel are in the open configuration to the relaxed unliganded state where the channel is closed have been described (Černý et al. 2019). The role of the 'clamshell' motion of the ligand binding domain (LBD) lobes in the structural transition is supplemented by the observed structural similarity at the level of protein domains during the structural transition, combined with the overall large rearrangement necessary for the opening and closing of the receptor. The activated and open states of the receptor are structurally similar to the liganded crystal structure, while in the unliganded receptor, the extracellular domains perform rearrangements leading to a clockwise rotation of up to 45 degrees around the longitudinal axis of the receptor, which closes the ion channel. The ligand-induced rotation of extracellular domains transferred by LBD-TMS linkers to the membrane-anchored ion channel is responsible for the opening and closing of the transmembrane ion channel (Černý et al. 2019).
Each subunit may span the membrane three times with the N-termini (the glutamate-binding domains) localized extracellularly and the C-termini localized cytoplasmically (Gouaux, 2004). The extracellular amino terminal domain, S1, and the loop domain between TMSs 2 and 3, bind the neurotransmitter (Gouaux, 2004). Between TMSs 1 and 2 is a P-loop which participates in channel formation and ion selectivity. Transmembrane AMPA receptor regulatory proteins and cornichons allosterically regulate AMPA receptor antagonists and potentiators (Schober et al., 2011; Coombs et al., 2012; Kato et al., 2010). The 3-d structure of a hetrotetrameric NMDA receptor/ion channel, GluN12GluN22, has been solved to 4 Å resolution (Karakas and Furukawa 2014). It is a symmetrical dimer of heterodimers. Channelopathies associate with abnormal gating pore mechanisms in GIC channels have been reviewed (Moreau et al. 2015). Mutations affecting structural equilibrium between cleft-locked and cleft-partially-open conformations have been described (Sakakura et al. 2019). The substitution-induced population shift in this equilibrium may be related to slower desensitization observed for these variants.
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 (Mayer, 2011). By contrast, the GluR 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 GluR 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 (Mayer, 2011).
The Mammalian ionotropic glutamate receptors (18 proteins) regulate a broad spectrum of processes in the brain, spinal cord, retina, and peripheral nervous system. They play important roles in numerous neurological diseases. They have multiple semiautonomous extracellular domains linked to a pore-forming element with striking resemblance to an inverted potassium channel. Traynelis et al. (2010) discussed glutamate receptor nomenclature, structure, assembly, accessory subunits, interacting proteins, gene expression and translation, post-translational modifications, agonist and antagonist pharmacology, allosteric modulation, mechanisms of gating and permeation, roles in normal physiological function, and the potential therapeutic use of pharmacological agents acting at glutamate receptors.
The subunits of GIC family channel proteins fall into six subfamilies: α, β, γ, δ, ε and ζ. Two regions in the N-terminal domain of glutamate receptor 3 form the subunit oligomerization interface that controls subtype-specific receptor assembly (Ayalon et al., 2005). The canonical conformational states occupied by most ligand-gated ion channels, and many cell-surface receptors, are the resting, activated, and desensitized states. The AMPA-sensitive GluR2 receptor undergoes conformational rearrangements of the agonist binding cores that occur upon desensitization. Desensitization involves the rupture of an extensive interface between domain 1 of 2-fold related glutamate-binding core subunits, compensating for the ca. 21 degrees of domain closure induced by glutamate binding. The rupture of the domain 1 interface allows the ion channel to close and thereby provides a simple explanation to the long-standing question of how agonist binding is decoupled from ion channel gating upon receptor desensitization (Armstrong et al., 2006). Auxiliary subunits have been described (Yan and Tomita, 2012).
The GIC channels are divided into three types: (1) α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)-, (2) kainate- and (3) N-methyl-D-aspartate (NMDA)-selective glutamate receptors. Subunits of the AMPA and kainate classes exhibit 35-40% identity with each other while subunits of the NMDA receptors exhibit 22-24% identity with the former subunits. They possess large N-terminal, extracellular glutamate-binding domains that are homologous to the periplasmic glutamine and glutamate receptors (TC #3.A.1.3.2 and TC #3.A.1.3.4, respectively) of ABC-type uptake permeases (TC #3.A.1) of Gram-negative bacteria. All functionally characterized members of the GIC family are from animals. The different channel (receptor) types exhibit distinct ion selectivities and conductance properties. The NMDA-selective large conductance channels are highly permeable to monovalent cations and Ca2+. The AMPA- and kainate-selective ion channels are permeable primarily to monovalent cations with only low permeability to Ca2+.
A prokaryotic K+-selective glutamate receptor that binds glutamate and forms K+-selective ion channels has been characterized (Chen et al., 1999). It shows sequence similarity to both glutamate receptors of eukaryotes and to K+ channels of the VIC family (TC #1.A.1). It exhibits 397 amino acyl residues, a signal peptide, and three TMSs flanked by two regions of about 140 residues. It showed highest sequence similarity to the rat δ1 GluR followed by a putative GluR from Arabidopsis thaliana. As a result of these observations, it has been proposed that glutamate receptors of eukaryotes arose from a primordial prokaryotic protein (Chen et al., 1999).
High-resolution structures of the ligand binding core of GluR0, a glutamate receptor ion channel from Synechocystis PCC 6803, have been solved by X-ray diffraction (Mayer et al., 2001). The GluR0 structures reveal homology with bacterial periplasmic binding proteins and the rat GluR2 AMPA subtype neurotransmitter receptor. The ligand binding site is formed by a cleft between two globular alpha/beta domains. L-Glutamate binds in an extended conformation, similar to that observed for glutamine binding protein (GlnBP). However, the L-glutamate γ-carboxyl group interacts exclusively with Asn51 in domain 1, different from the interactions of ligand with domain 2 residues observed for GluR2 and GlnBP. To address how neutral amino acids activate GluR0 gating, Mayer et al. (2001) solved the structure of the binding site complex with L-serine. This revealed solvent molecules acting as surrogate ligand atoms, such that the serine OH group makes solvent-mediated hydrogen bonds with Asn51. The structure of a ligand-free, closed-cleft conformation revealed an extensive hydrogen bond network mediated by solvent molecules. Equilibrium centrifugation analysis revealed dimerization of the GluR0 ligand binding core with a dissociation constant of 0.8 microM. In the crystal, a symmetrical dimer involving residues in domain 1 occurs along a crystallographic 2-fold axis and suggests that tetrameric glutamate receptor ion channels are assembled from dimers of dimers. They propose that ligand-induced conformational changes cause the ion channel to open as a result of an increase in domain 2 separation relative to the dimer interface.
Ionotropic glutamate receptor (GluR) ion channels share structural similarity, albeit an inverted membrane topology, with P-loop channels. Like P-loop channels, prokaryotic GluR subunits (e.g. GluR0 of Synechocystis (TC# 1.A.10.2.1)) have two transmembrane channel-forming segments. In contrast, eukaryotic GluRs have an additional transmembrane segment (M4), located C-terminal to the ion channel core. Although topologically similar to GluR0, mammalian AMPA receptor (GluA1) subunits lacking the M4 segment do not display surface expression. In the AMPA receptor structure, a face in M4 forms intersubunit contacts with the transmembrane helices of the ion channel core (M1 and M3) from another subunit within the homotetramer. Thus, a highly specific interaction of the M4 segment with an adjacent subunit is required for surface expression of AMPA receptors (Salussolia et al., 2011).
AMPA receptors are homo or heterooligomers of four subunits, GluRA-D (also called GluR1-4). The GluRB subunit of the AMPA receptor, responsible for fast excitatory signaling in the brain and ion selectivity, has been purified in milligram quantities as a homotetramer. It exhibits the expected pharmacological properties. Based on molecular mass and electron microscopic studies, the channel appears to be a dimer of dimers (Safferling et al., 2001). The molecular dimensions are about 11 x 14 x 17 nm, and solvent accessible regions that may form the channel can be seen. AMPA channels are regluated by transmembrane AMPA receptor regulatory proteins (TARPs) which exert their effects principally on the channel opening reaction. A thermodynamic argument suggests that because TARPs promote channel opening, receptor activation promotes AMPAR-TARP complexes into a superactive state with high open probability (Carbone and Plested 2016).
Ligand (neurotransmitter) binding opens the transmembrane pore, but after activation, desensitization results, in which the ligand remains bound, but the ion channel is closed. Using the GluR2 AMPA-sensitive glutamate receptor, Sun et al. (2002) showed (1) that the ligand-binding cores form the dimer interfaces, (2) that stabilization of the intradimer interface reduces desensitization, (3) that destabilization of the intradimer interface enhances desensitization, and (4) receptor activation involves conformational changes within each subunit that result in an increase in the separation of portions of the receptor that are linked to the channel. These results indicate how ligand binding is coupled to channel activation (gating), suggest modes of dimer-dimer interaction in the formation of the tetramer, and show that desensitization results from rearrangement of the dimer interface which disengages the agonist-induced conformational change in the ligand-binding core from the ion channel gate (Sun et al., 2002).
NMDA receptors are always heterotetrameric cation channels that transport Ca2+ with subunits NR1, NR2 and NR3 in an (NR1)2 (NR2)2 or (NR1)2 (NR2)(NR3) arrangement (Furukawa et al., 2005). Glycine binds to NR1, and glutamate binds to NR2 and/or NR3, and simultaneous binding of both agonists as well as relief of Mg2+ blockage by membrane depolarization is required for channel opening. There are four types of auxillary subunits for iGluRs. They are calledTARPs, cornichons, neuropilins and tolloid-like proteins (NETO). They and their descriptions can be found in TC families 8.A.16 and 8.A.47.
Crystal structures of the ligand binding core of NR2A with glutamate and of the NR1-NR2A heterodimer with glycine and glutamate bound. The details of subunit:subunit interaction and of channel opening were reported (Furukawa et al., 2005). As a result, many features including the mechanism of allosteric modulation of channel activity (Jin et al., 2005) and the mechanism of dual agonist action (Olson and Gouaux, 2005) were revealed.
Glutamate receptor ligand binding domain dimer assembly is modulated allosterically by ions (Chaudhry et al., 2009). The activities of many ligand-gated ion channels and cell surface receptors are modulated by small molecules and ions. For kainate, but not AMPA subtype glutamate receptors, the binding of Na+ and Cl- ions to discrete, electrostatically coupled sites in the extracellular ligand binding domain (LBD), regulates dimer assembly. Dimer assembly then regulates the rate of entry into the desensitized state, which occurs when the dimer interface ruptures and the channel closes. Studies on glutamate receptors have defined the LBD dimer assembly as a key functional unit that controls activation and desensitization. Sodium and chloride ions modulate kainate receptor dimer affinity as much as 50-fold, and removal of either Cl- or Na+ disrupts the dimer (Chaudhry et al., 2009).
Ionotropic glutamate receptors (iGluRs) mediate fast excitatory synaptic transmission in the central nervous system. Upon agonist binding, an iGluR opens to allow the flow of cations and subsequently enters into a desensitized state. Dong and Zhou (2011) reported molecular dynamics simulations of an AMPA-subtype iGluR Channel opening and closing were observed in simulations of the activation and desensitization processes, respectively. The motions of the LBD-TMD linkers along the central axis of the receptor and in the lateral plane contributed cooperatively to channel opening and closing. Transmembrane AMPAR regulatory protein (TARP) gamma-7 (TC#8.A.16.2.5) selectively enhances the synaptic expression of Ca2+-permeable (CP-AMPARs) and suppresses calcium-impermeable (CI-AMPAR) activities (Studniarczyk et al. 2013). Thus, TARPs modulate the pharmacology and gating of AMPA-type glutamate receptors (Soto et al. 2014). TARPs interact with the N-terminal domain of the AMPARs and control channel gating; residues in the receptor and the TARP involved in this interaction have been identified (Cais et al. 2014). Glutamatergic mechanisms related to epilepsy have been reviewed by Dingledine (2012).
Cys loop, glutamate, and P2X receptors are ligand-gated ion channels (LGICs) with 5, 4, and 3 protomers, respectively. Agonists and competitive antagonists apparently induce opposite motions of the binding pocket (Du et al., 2012). Agonists, usually small, induce closure of the binding pocket, leading to opening of the channel pore, whereas antagonists, usually large, induce opening of the binding pocket, thereby stabilizing the closed pore.
AMPA receptors (AMPAR) are the main ligand-gated ion channels responsible for the fast excitatory synaptic transmission in the mammalian brain. A number of proteins that interact with AMPAR are known to be involved in the trafficking and localization of the receptor and/or the regulation of receptor channel properties. Additionally, the presence of up to 34 proteins may interact as high-confidence constituents of the AMPAR. The inner core of the receptor complex may consist of the GluA tetramer and four auxiliary proteins comprising transmembrane AMPA receptor regulatory proteins and/or cornichons. The other AMPAR interactors, present in lower amount, may form the outer shell of the AMPAR with a range in size and variability (Li et al. 2013).
Ionotropic glutamate receptors comprise two conformationally different A/C and B/D subunit pairs. Closed channels exhibit fourfold radial symmetry in the transmembrane domain (TMD) but transition to twofold dimer-of-dimers symmetry for extracellular ligand binding and N-terminal domains. It has been suggested that fourfold pore symmetry persists in the open state (Wilding et al. 2014).
As noted above, N-Methyl-D-aspartate (NMDA) receptors belong to the family of ionotropic glutamate receptors, which mediate most excitatory synaptic transmission in mammalian brains. Calcium permeation triggered by activation of NMDA receptors is the pivotal event for initiation of neuronal plasticity. Karakas and Furukawa 2014 determined the crystal structure of the intact heterotetrameric GluN1-GluN2B NMDA receptor ion channel at 4 angstroms. The NMDA receptors are arranged as a dimer of GluN1-GluN2B heterodimers with the twofold symmetry axis running through the entire molecule composed of an amino terminal domain (ATD), a ligand-binding domain (LBD), and a transmembrane domain (TMD). The ATD and LBD are much more highly packed in the NMDA receptors than non-NMDA receptors, which may explain why ATD regulates ion channel activity in NMDA receptors but not in non-NMDA receptors (Karakas and Furukawa 2014).
iGluRs include AMPA receptor (AMPAR) and NMDA receptor (NMDAR)subtypes. The iGluR pore domain is structurally and evolutionarily related to an inverted two-transmembrane K+ channel. Peripheral to the pore domain in eukaryotic iGluRs is an additional transmembrane helix, the M4 segment, which interacts with the pore domain of a neighboring subunit. In AMPARs, the integrity of the alignment of a specific face of M4 with the adjacent pore domain is essential for receptor oligomerization. In contrast to AMPARs, NMDARs are obligate heterotetramers composed of two GluN1 and typically two GluN2 subunits. Although the AMPAR M4 contributes minimally to receptor desensitization, the NMDAR M4 segments have robust and subunit-specific effects on desensitization. Thus, the functional roles of the M4 segments in AMPARs and NMDARs are different, and the M4 segments in NMDARsmay provide a transduction pathway for receptor modulation at synapses (Amin et al. 2017).
Pang and Zhou 2017 reported the structural modeling for the open state of an NMDA receptor. Staring from the crystal structure of the closed state, they repacked the pore-lining helices to generate an initial open model. This model was modified to ensure tight packing between subunits and then refined by a molecular dynamics simulation in explicit membrane. They identified Cα-H...O hydrogen bonds between the Cα of a conserved glycine in one transmembrane helix and a carbonyl oxygen of a membrane-parallel helix at the extracellular side of the transmembrane domain as important for stabilizing the open state. This observation may explain why mutations of this glycine are associated with neurological diseases that lead to significant decreases in channel open probability (Pang and Zhou 2017).
AMPA receptors coassemble with transmembrane AMPA receptor regulatory proteins (TARPs), yielding a receptor complex with altered gating kinetics, pharmacology, and pore properties. Chen et al. 2017 elucidated structures of the GluA2-TARP gamma2 complex in the presence of the partial agonist kainate or the full agonist quisqualate together with a positive allosteric modulator or with quisqualate alone. They showed how TARPs sculpt the ligand-binding domain gating ring, enhancing kainate potency and diminishing the ensemble of desensitized states. TARPs encircle the receptor ion channel, stabilizing M2 helices and pore loops, illustrating how TARPs alter receptor pore properties. Structural and computational analyses suggested that the full agonist and modulator complex harbors an ion-permeable channel gate, providing the first view of an activated AMPA receptor (Chen et al. 2017). AMPA receptors co-assemble with auxiliary proteins, such as stargazin, which can markedly alter receptor trafficking and gating. Stargazin acts in part to stabilize or select conformational states that favor activation (Shaikh et al. 2016).
N-methyl-D-aspartate receptors (NMDARs) mediate excitatory synaptic transmission in the central nervous system and underlie the induction of synaptic plasticity; their malfunction is associated with human diseases. Native NMDARs are tetramers composed of two obligatory GluN1 subunits and various combinations of GluN2A-D or, more rarely, GluN3A-B subunits. Each subunit consists of amino-terminal, ligand-binding, transmembrane and carboxyl-terminal domains. The ligand-binding and transmembrane domains are interconnected via linkers. Upon glutamate or glycine binding, these receptors undergo a series of conformational changes, opening the Ca2+-permeable ion channel. Ladislav et al. 2018 reported that different deletions and mutations of residues in the M3-S2 linkers of the GluN1 and GluN2B subunits lead to constitutively open channels. Irrespective of whether alterations were introduced in the GluN1 or the GluN2B subunit, application of glutamate or glycine promoted receptor channel activity; however, responses induced by the GluN1 (Ladislav et al. 2018).
Impaired hippocampal synaptic plasticity contributes to cognitive impairment in Huntington's disease (HD). AMPAR surface diffusion, a key player in synaptic plasticity, is disturbed in various rodent models of HD. Zhang et al. 2018 demonstrated that defects in the brain-derived neurotrophic factor (BDNF)-tyrosine receptor kinase B (TrkB) signaling pathway contribute to the deregulated AMPAR trafficking by reducing the interaction between transmembrane AMPA receptor regulatory proteins (TARPs, TC# 8.A.16.2) and the PDZ-domain scaffold protein PSD95 (TC# 8.A.24.1.3). The disturbed AMPAR surface diffusion is rescued by the antidepressant drug tianeptine via the BDNF signaling pathway. Tianeptine also restores the impaired LTP and hippocampus-dependent memory in different HD mouse models. These findings unravel a mechanism underlying hippocampal synaptic and memory dysfunction in HD, and highlight AMPAR surface diffusion as a promising therapeutic target (Zhang et al. 2018).
Homotetrameric AMPA receptor channels open in a stepwise manner, consistent with independent activation of individual subunits, and they exhibit complex kinetic behavior that manifests as temporal shifts between four different conductance levels. Shi et al. 2019 investigated how two AMPA receptor-selective noncompetitive antagonists disrupt the intrinsic step-like gating patterns of maximally activated homotetrameric GluA3 receptors. Interactions of 2,3-benzodiazepines with residues in the boundary between the extracellular linkers and transmembrane helical domains reorganize the gating behavior of channels. Low concentrations of modulators stabilize open and closed states to different degrees and coordinate the activation of subunits so that channels open directly from closed to higher conductance levels. Using kinetic and structural models, Shi et al. 2019 provided insight into how the altered gating patterns might arise from molecular contacts within the extracellular linker-channel boundary.
Glutamate-gated AMPA receptors mediate the fast component of excitatory signal transduction at chemical synapses throughout all regions of the mammalian brain. AMPA receptors are tetrameric assemblies composed of four subunits, GluA1-GluA4. Zhao et al. 2019 elucidated the structures of 10 distinct native AMPA receptor complexes by single-particle cryo-EM. They found that receptor subunits are arranged nonstochastically, with the GluA2 subunit preferentially occupying the B and D positions of the tetramer and with triheteromeric assemblies comprising a major population of native AMPA receptors. Cryo-EM maps defined the structure for S2-M4 linkers between the ligand-binding and transmembrane domains, suggesting how neurotransmitter binding is coupled to ion channel gating (Zhao et al. 2019).
The generalized transport reaction catalyzed by GIC family channels is:
Me+ (or Me2+) (out) ⇌ Me+ (or Me2+) (in).
AMPA-selective glutamate ionotropic channel receptor (GIC; AMPAR), kainate-subtype, GluR-K1; GluR1; GluR-A; GluA1; Gria1 of 906 aas (preferentially monovalent cation selective). Contributes to amygdala-dependent emotional learning and fear conditioning (Humeau et al., 2007). Transmembrane AMPAR regulatory protein (TARP) gamma-7 (TC#8.A.16.2.5) selectively enhances the synaptic expression of Ca2+-permeable (CP-AMPARs) and suppresses calcium-impermeable (CI-AMPAR) activities (Studniarczyk et al. 2013). Thus, TARPs modulate the pharmacology and gating of AMPA-type glutamate receptors (Soto et al. 2014). TARPs interact with the N-terminal domain of the AMPAR and control channel gating; residues in the receptor and the TARP involved in this interaction have been identified (Cais et al. 2014). The auxilary protein, Shisa9 or CKAMP44 (UniProt acc# B4DS77), has a C-terminal PDZ domain that allows interaction with scaffolding proteins and AMPA glutamate receptors (Karataeva et al. 2014). The transmembrane domain alone can tetramerize (Gan et al. 2016). The most potent and well-tolerated AMPA receptor inhibitors, used to treat epilepsy, act via a noncompetitive mechanism. The crystal structures of the rat AMPA-subtype GluA2 receptor in complex with three noncompetitive inhibitors have been solved. The inhibitors bind to a binding site, completely conserved between rat and human, at the interface between the ion channel and linkers connecting it to the ligand-binding domains (Yelshanskaya et al. 2016). The endogenous neuropeptide, cyclopropylglycine, at a physiological concentration of 1 μM, enhances the transmembrane AMPA currents in rat cerebellar Purkinje cells (Gudasheva et al. 2016). The energetics of glutamate binding have been estimated (Yu and Lau 2017). The TMEM108 protein (Q6UXF1 of 575 aas and 2 TMSs, N- and C-terminal, is required for surface expression of AMPA receptors (Jiao et al. 2017). CERC-611 is a selective antagonist of AMPA receptors containing transmembrane AMPA receptor regulatory protein (TARP; TC# 8.A.16) gamma-8 (Witkin et al. 2017).
GluR-K1 of Rattus norvegicus
The homo- and heteromeric glutamate receptor, GLR3.3/3.4 (Desensitized in 3 patterns: (1) by Glu alone; (2) by Ala, Cys, Glu or Gly; (3) by Ala, Cys, Glu, Gly, Ser or Asn (Stephens et al., 2008). A regulatory mechanism underlies Ca2+ homeostasis by sorting and activation of AtGLRs by AtCNIHs (see for example, 8.A.61.1.9) (Wudick et al. 2018). May be responsible in part for Cd2+ uptake (Chen et al. 2018). GLR3.3 and GLC3.6 (TC# 1.A.10.1.24) (but not GLR3.4) play different roles in nervous system-like signaling in plant defense by a mechanism that differs substantially from that in animals (Toyota et al. 2018).
GLR3.3/GLR3.4 receptor of Arabidopsis thaliana
GriK2; GluK2; GluR6 glutamate receptor, ionotropic kainate 2. The 3-d structure is known (2XXY_A). The domain organization and function have been analyzed by Das et al. (2010). Two auxiliary subunits, Neto1 and Neto2 (Neuropilin and tolloid-like proteins) alter the trafficking, channel kinetics and pharmacology of the receptors (Howe 2014). They reduce inward rectification without altering calcium permeability (Fisher and Mott 2012). Interactions between the pore helix (M2) and adjacent segments of the transmembrane inner (M3) and outer (M1) helices may be involved in gating (Lopez et al. 2013). Mutations in the human GRIK2 (GLUR6) cause moderate-to-severe nonsyndromic autosomal recessive mental retardation (Motazacker et al. 2007). Kainate receptors regulate KCC2 (TC# 1.A.10.1.11) expression in the hippocampus (Pressey et al. 2017). GluR6, carrying the pore loop plus adjacent transmembrane domains of the prokaryotic, glutamate-gated, K+-selective GluR0 (TC# 1.A.10.2.1), adopted several electrophysiological properties of the donor pore uponpore transplantation (Hoffmann et al. 2006).
Grik2 of Rattus norvegicus (P42260)
The NMDA receptor. The crystal structure of the N-terminal domains (GluN1 and GluN2) have been determined (PDB#3QEL; Talukder and Wollmuth, 2011). The ligand-specific deactivation time courses of GluN1/GluN2D NMDA receptors have been measured (Vance et al., 2011). NMDA receptors are Hebbian-like coincidence detectors, requiring binding of glycine and glutamate in combination with the relief of voltage-dependent magnesium block to open an ion conductive pore. Lee et al. 2014 presented X-ray structures of the Xenopus laevis GluN1-GluN2B NMDA receptor with the allosteric inhibitor, Ro25-6981, partial agonists and the ion channel blocker, MK-801. Receptor subunits are arranged in a 1-2-1-2 fashion, demonstrating extensive interactions between the amino-terminal and ligand-binding domains. The 3-TMS transmembrane domains harbour a closed-blocked ion channel, a pyramidal central vestibule lined by residues implicated in binding ion channel blockers and magnesium, and a approximately twofold symmetric arrangement of ion channel pore loops. GRIN2D mediates developmental and epileptic encephalopathy (XiangWei et al. 2019).
NMDA receptor of Xenopus laevis (Q91977)
Glu2 AMPA receptor (GluR-2; GluR2-flop; CX614; GluA2). The 3-d structure is known at 3.6 Å resolution. It shows a 4-fold axis of symmetry in the transmembrane domain, and a 2-fold axis of symmetry overall, although it is a homotetramer (Sobolevsky et al. 2009). A structure showing an agoniar-bound form of the rat GluA2 receptor revealed conformational changes that occur during gating (Yelshanskaya et al. 2014). GluR2 interacts directly with β3 integrin (Pozo et al., 2012). In general, integrin receptors form macromolelcular complexes with ion channels (Becchetti et al. 2010). TARPS are required for AMP receptor function and trafficking, but seven other auxiliary subunits have also been identified (Sumioka 2013). For example, AMPA receptors are regulated by S-SCAM through TARPs (Danielson et al. 2012). The C-terminal domains of various TARPs (TC#8.A.16.2) play direct roles in the regulation of GluRs (Sager et al. 2011). Whole-genome analyses have linked multiple TARP loci to childhood epilepsy, schizophrenia and bipolar disorders (Kato et al. 2010). Thus, TARPs emerge as vital components of excitatory synapses that participate both in signal transduction and in neuropsychiatric disorders. The architecture of a fully occupied GluR2-TARP complex has been illucidated by cryoEM, showing the homomeric GluA2 AMPA receptor saturated with TARP Υ2 subunits, showing how the TARPs are arranged with four-fold symmetry around the ion channel domain, making extensive interactions with the M1, M2 and M4 TMSs (Zhao et al. 2016). The binding mode and sites for prototypical negative allosteric modulators at the GluA2 AMPA receptor revealing new details of the molecular basis of molulator binding and mechanisms of action (Stenum-Berg et al. 2019).
GluR-2 of Homo sapiens (P42262)
Ionotropic receptor 25a, Ir25a. Not involved in salt sensing (Zhang et al. 2013). It resets the circadian clock in response to temperature (Chen et al. 2015).
Ir25a of Drosophila melanogaster
Glutamate ionotropic receptor homologue
Invertebrate animals (insects)
Glutamate receptor in Daphnia pulex (water flea)
Olfactory ionotropic receptor, Ir93a of 842 aas
Ir93a of Panulirus argus (spiny lobster)
Ionotropic sodium channel; attractive, sodium gustatory sensory receptor for positive salt taste. Not involved in salt avoidance which uses a distinct receptor (Zhang et al. 2013).
Ir76b of Drosophila melanogaster
Calcium channel of 551 aas, Glr1 (Wheeler and Brownlee 2008).
Glr1 of Chlamydomonas reinhardtii
Olfactory glutamate-like ionotropic receptor, kainate 2 isoform X1 of 754 aas and 4 TMSs. Chen et al. 2017 identify 102 putative IR genes, (dubbed AalbIr genes) in the mosquito Aedes albopictus (Skuse), 19 of which showed expression in the female antenna. These putative olfactory AalbIRs share four conservative hydrophobic domains similar to the transmembrane and ion channel pore regions found in conventional iGluRs. To determine their potential functions in host-seeking, Chen et al. 2017 compared their transcript expression levels in the antennae of blood-fed females with that of non-blood-fed females. Three AalbIr genes showed downregulation when the mosquito finished a bloodmeal.
Olfactory receptor of Aedes albopictus (Asian tiger mosquito) (Stegomyia albopicta)
Glutamate receptor 4, GIC, AMPA-subtype, GluR4, GRIA4 or GluR-D (preferentially monovalent cation selective). Binding of the excitatory neurotransmitter, L-glutamate, induces a conformation change, leading to the opening of the cation channel, thereby converting the chemical signal to an electrical impulse. The receptor then desensitizes rapidly and enters a transient inactive state, characterized by the presence of bound agonist. In the presence of CACNG4, CACNG7 or CACNG8, GluR4 shows resensitization characterized by a delayed accumulation of current flux upon continued application of glutamate (Gill et al. 2008; Birdsey-Benson et al. 2010). De novo variants in GRIA4 lead to intellectual disability with or without seizures, gait abnormalities, problems of social behavior, and other variable features (Martin et al. 2017).
GluR-D of Rattus norvegicus
Heteromeric ionotropic NMDA receptor (NMDAR) consisting of two subunits, GluN1 (938 aas) and GluN2A (1464 aas). Positions of the Mg2+ and Ca2+ ions in the ion channel divalent cation binding site have been proposed, and differences in the structural and dynamic behavior of the channel proteins in the presence of Mg2+ or Ca2+ have been analyzed (Mesbahi-Vasey et al. 2017). GRIN variants in receptor M2 channel pore-forming loop are associated with neurological diseases (Li et al. 2019)
NMDAR of Homo sapiens
Glutamate receptor 1, GluR1; Glr-1 of 962 aas and 5 TMSs. Plays a role in controlling movement in
response to environmental cues such as food availability and
mechanosensory stimulation such as the nose touch response (Campbell et al. 2016). Regluated by SOL1 (TC# 8.A.47.2.1) (Walker et al. 2006).
Glr-1 of Caenorhabditis elegans
NMDA-like glutamate receptor, NR1, of 964 aas and 4 TMSs. It functions in the organization of feeding, locomotory and defensive behaviors. Two are present, NR1-1 and NR1-2 in nurrons (Ha et al. 2006).
NR1 of Aplysia californica (California sea hare)
Ionotropic glutamate receptor, GluR1 (GluR-1, GluR1-flip; GRIA1; GluH1; CTZ) of 906 aas and 4 - 6 TMSs. L-glutamate acts as an excitatory neurotransmitter at many synapses in the central nervous system. Binding of the excitatory neurotransmitter, L-glutamate, induces a conformational change, leading to the opening of the cation-specific channel, thereby converting the chemical signal to an electrical impulse upon entry of Na+ and Ca2+. The receptor then desensitizes rapidly and enters a transient inactive state, characterized by the presence of bound agonist. In the presence of CACNG4 or CACNG7 or CACNG8, it shows resensitization characterized by a delayed accumulation of current flux upon continued application of glutamate (Kato et al. 2010). The polyamines, spermine, spermidine and putrescine can be drawn into the permeation pathway and get stuck, blocking the movement of other ions. The degree of this polyamine-mediated channel block is highly regulated by processes that control the free cytoplasmic polyamine concentration, the membrane potential, and the iGluR subunit composition (Bowie 2018).
GluR-1 of Homo sapiens
Glutamate-gated receptor 3.6 of 903 aas, GLR3.6. It probably acts as non-selective cation channel, transporting Ca2+ into the cell. It mediates leaf-to-leaf wound signaling. GLR3/6 may be involved in light-signal transduction and calcium homeostasis via the regulation of calcium influx into cells (Mousavi et al. 2013). Together with GLR3.3 (TC# 1.A.10.1.10), it plays a roles in nervous system-like signaling in plant defense. GLR3.3 and GLR3.6 play different roles by a mechanism that differs substantially from that in animals (Toyota et al. 2018).
GLR3.6 of Arabidopsis thaliana
GIC, NMDA-subtype, Grin C2 (highly permeable to Ca2+ and monovalent cations). A single residue in the GluN2 subunit controls NMDA receptor channel properties via intersubunit interactions (Retchless et al., 2012). Memantine (Namenda) is prescribed as a treatment for moderate to severe Alzheimer's Disease. Memantine functions by blocking the NMDA receptor, and the sites of interaction have been identified (Limapichat et al. 2013). Genetic mutations in multiple NMDAR subunits cause various childhood epilepsy syndromes (Li et al. 2016). NMDA receptor gating is complex, exhibiting multiple closed, open, and desensitized states, but the structure-energy landscape of gating for the rat homologue has been mapped (Dolino et al. 2017). NMDARs are tetrameric complexes consisting of two glycine-binding GluN1 and two glutamate-binding GluN2 subunits. Four GluN2 subunits encoded by different genes can produce up to ten different di- and triheteromeric receptors. These heteromeric systems have been modeled (Gibb et al. 2018). A conserved glycine associated with diseases permits NMDA receptors to acquire high Ca2+ permeability (Amin et al. 2018). The ND2 protein (see TC# 3.D.1.6.1), a component of the NMDAR complex, enables Src tyrosine protein kinase (TC# 8.A.23.1.12) regulation of NMDA receptors (Scanlon et al. 2017).
NMDA receptor, Grin C2, of Homo sapiens
AMPA glutamate receptor 3 (GluR3, GluA3. GRIA3. LLUR3. GLURC) (non-selective monovalent cation channel and Ca2+ channel) (Ayalon et al., 2005; Midgett et al., 2012). Regulated by AMPA receptor regulatory proteins (TARPs) including stargazin and CNIH auxiliary subunits (Kim et al., 2010; Straub and Tomita, 2011; Jackson and Nicoll, 2011; Bats et al., 2012; Rigby et al. 2015). The domain architecture of a calcium-permeable AMPA receptor in a ligand-free conformation has been solved (Midgett et al., 2012). The TARP, stargazin, is elevated in the somatosensory cortex of Genetic Absence Epilepsy Rats (Kennard et al. 2011). TARPs alter the conformation of pore-forming subunits and thereby affect antagonist interactions (Cokić and Stein 2008). The structural basis of AMPAR regulation by TARP gamma2, or stargazin (STZ) involves variable interaction stoichiometries of the AMPAR-TARP complex, with one or two TARP molecules binding one tetrameric AMPAR (Twomey et al. 2016). The ion channel extracellular collar plays a role in gating and represents a hub for powerful allosteric modulation of AMPA receptor function (Yelshanskaya et al. 2017). The A653T mutation stabilizes the closed configuration of the channel and affects duration of sleep and awake periods in both humans and mice (Davies et al. 2017). The tetramer exhibits 4 distinct conductase leves due to independent subunit activation. Perampanel is an anticonvulsant drug that regulates gating (Yuan et al. 2019).
GluR3 of Homo sapiens (P42263)
The homomeric cation channel/glutamate receptor/kainate 1, GluR5 (weakly responsive to glutamate) (expressed in the developing nervous system) (Bettler et al., 1990). The 3-d structures of the protein have been determined with agonists and antagonists. The agonist, domoic acid, stabilizes the ligand-binding core of the iGluR5 complex in a conformation that is 11 degrees more open than the conformation observed when the full agonist, (S)-glutamate, is bound (Hald et al. 2007). Kainate receptors regulate KCC2 expression in the hippocampus (Pressey et al. 2017).
GluR5 of Rattus norvegicus
The heteromeric monovalent cation/Ca2+ channel/glutamate (NMDA) receptor NMDAR1/NMDAR2A/NMDAR2B/NMDAR2C) (Monyer et al., 1992). Note: NR2B is the same as NR3, GluN2A, GRIN2A or subunit epsilon (Schüler et al., 2008). Mediates voltage- and Mg2+-dependent control of Na+ and Ca2+ permeability (Yang et al., 2010). Mutations in the subunit, GRIN1, a 1464 aa protein, identified in patients with early-onset epileptic encephalopathy and profound developmental delay, are located in the transmembrane domain and the linker region between the ligand-binding and transmembrane domains (Yuan et al. 2014; Ohba et al. 2015). Karakas and Furukawa 2014 determined the crystal structure of the heterotetrameric GluN1-GluN2B NMDA receptor ion channel at 4 Å resolution. The receptor is arranged as a dimer of GluN1-GluN2B heterodimers with the twofold symmetry axis running through the entire molecule composed of an amino terminal domain, a ligand-binding domain, and a transmembrane domain. The GluN2 subunit regulates synaptic trafficking of AMPA in the neonatal mouse brain (Hamada et al. 2014). GRIN1 and GRIN2A mutations are associated with severe intellectual disability with cortical visual impairment, epilepsy and oculomotor and movement disorders being discriminating phenotypic features (Lemke et al. 2016; Chen et al. 2017).The cryoEM structure of a triheteromeric receptor including GluN1 (glycine binding), GluN2A and GluN2B (both glutamate binding)has been solved with and without a GluN2B the allosteric antagonist, Ro 25-6981 (Lü et al. 2017). Ogden et al. 2017 implicated the pre-M1 region in gating, provided insight into how different subunits contribute to gating, and suggested that mutations in the pre-M1 helix, such as those that cause epilepsy and developmental delays, can compromise neuronal health. The severity of GRIN2A (Glu2A)-related disorders can be predicted based on the positions of the mutations in the encoding gene (Strehlow et al. 2019).
NR1/NR2A or NR2B or NR2C of Rattus norvegicus
The mouse glutamate receptor δ-2 subunit precursor (GluR δ-2, GluR delta subunit, or GluD2) (Uemura et al., 2004). The 3-d structure in the synaptic junctional complex with presynaptic β-neurexin 1 (β-NRX1 or NRXN1A; Q9ULB1 = the human homologue) and the C1q-like synaptic organizer, cerebellin-1 (Cbln1; 193 aas, 1 or 2 TMSs; Q9R171 = the human homolgue) has been solved (Elegheert et al. 2016).
GluR δ2 of Mus musculus (Q61625)
Glutamate-gated ionotropic K+ channel receptor, GluR0 (5TMSs). X-ray structures are available (PDB: 1IIT) (Lee et al. 2005; Lee et al. 2008) GluR6 (TC# 1.A.10.1.11), carrying the pore loop plus adjacent transmembrane domains of this prokaryotic, glutamate-gated, K+-selective GluR0, adopted several electrophysiological properties of the donor pore upon pore transplantation (Hoffmann et al. 2006).
GluR0 of Synechocystis sp. PCC6803
Probable Ionotropic glutamate receptor (GluR)
GluR homologue of Algoriphagus sp. PR1 (A3I049)
Probably Ionotropic glutamate receptor (GluR)
GluR homologue of Chlorobium luteolum (Q3B5G3)
Probable Ionotropic glutamate receptor (GluR)
GluR homologue of Vibrio fischeri (B5FDH7)
Uncharacterized protein of 1003 aas and 5 - 7 TMSs
UP of Chlamydomonas reinhardtii (Chlamydomonas smithii)
Ionotropic ligand (glutamate) receptor of 433 aas and 3 or 4 TMSs (Greiner et al. 2018).
GluR of Paramecium bursaria Chlorella virus IL-3A