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G-protein receptors (e.g., TC# 9.A.14.15.3) share an ANF receptor family, ligand binding region/domain (M. Saier, unpublished observation).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).
Herguedas et al. 2019 presented a cryo-EM structure of the heteromeric GluA1/2 receptor associated with two transmembrane AMPAR regulatory protein (TARP) gamma8 auxiliary subunits, the principal AMPAR complex at hippocampal synapses. Within the receptor, the core subunits arrange to give the GluA2 subunit dominant control of gating. This structure reveals the geometry of the Q/R site that controls calcium flux, suggests association of TARP-stabilized lipids, and demonstrates that the extracellular loop of gamma8 modulates gating by selectively interacting with the GluA2 ligand-binding domain. Collectively, this structure provides a blueprint for deciphering the signal transduction mechanisms of synaptic AMPARs (Herguedas et al. 2019).
The generalized transport reaction catalyzed by GIC family channels is:
Me+ (or Me2+) (out) ⇌ Me+ (or Me2+) (in).