Kainate receptor channel opening and gating mechanism.

Kainate receptors, a subclass of ionotropic glutamate receptors, are tetrameric ligand-gated ion channels that mediate excitatory neurotransmission1-4. Kainate receptors modulate neuronal circuits and synaptic plasticity during the development and function of the central nervous system and are implicated in various neurological and psychiatric diseases, including epilepsy, depression, schizophrenia, anxiety and autism5-11. Although structures of kainate receptor domains and subunit assemblies are available12-18, the mechanism of kainate receptor gating remains poorly understood. Here we present cryo-electron microscopy structures of the kainate receptor GluK2 in the presence of the agonist glutamate and the positive allosteric modulators lectin concanavalin A and BPAM344. Concanavalin A and BPAM344 inhibit kainate receptor desensitization and prolong activation by acting as a spacer between the amino-terminal and ligand-binding domains and a stabilizer of the ligand-binding domain dimer interface, respectively. Channel opening involves the kinking of all four pore-forming M3 helices. Our structures reveal the molecular basis of kainate receptor gating, which could guide the development of drugs for treatment of neurological disorders.

Kainate receptor modulation by NETO2.

Glutamate-gated kainate receptors are ubiquitous in the central nervous system of vertebrates, mediate synaptic transmission at the postsynapse and modulate transmitter release at the presynapse1-7. In the brain, the trafficking, gating kinetics and pharmacology of kainate receptors are tightly regulated by neuropilin and tolloid-like (NETO) proteins8-11. Here we report cryo-electron microscopy structures of homotetrameric GluK2 in complex with NETO2 at inhibited and desensitized states, illustrating variable stoichiometry of GluK2-NETO2 complexes, with one or two NETO2 subunits associating with GluK2. We find that NETO2 accesses only two broad faces of kainate receptors, intermolecularly crosslinking the lower lobe of ATDA/C, the upper lobe of LBDB/D and the lower lobe of LBDA/C, illustrating how NETO2 regulates receptor-gating kinetics. The transmembrane helix of NETO2 is positioned proximal to the selectivity filter and competes with the amphiphilic H1 helix after M4 for interaction with an intracellular cap domain formed by the M1-M2 linkers of the receptor, revealing how rectification is regulated by NETO2.

Structural biology of kainate receptors.

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.

Structural dynamics of the GluK3-kainate receptor neurotransmitter binding domains revealed by cryo-EM.

Kainate receptors belong to the ionotropic glutamate receptor family and play critical roles in the regulation of synaptic networks. The kainate receptor subunit GluK3 has unique functional properties and contributes to presynaptic facilitation at the hippocampal mossy fiber synapses along with roles at the post-synapses. To gain structural insights into the unique functional properties and dynamics of GluK3 receptor, we imaged them via electron microscopy in the apo-state and in complex with either agonist kainate or antagonist UBP301. Our analysis of all the GluK3 full-length structures not only provides insights into the receptor transitions between desensitized and closed states but also reveals a "non-classical" conformation of neurotransmitter binding domain in the closed-state distinct from that observed in AMPA and other kainate receptor structures. We show by molecular dynamics simulations that Asp759 influences the stability of the LBD dimers and hence could be responsible for the observed conformational variability and dynamics of the GluK3 via electron microscopy. Lower dimer stability could explain faster desensitization and low agonist sensitivity of GluK3. In overview, our work helps to associate biochemistry and physiology of GluK3 receptors with their structural biology and offers structural insights into the unique functional properties of these atypical receptors.

Structural basis of kainate subtype glutamate receptor desensitization.

Glutamate receptors are ligand-gated tetrameric ion channels that mediate synaptic transmission in the central nervous system. They are instrumental in vertebrate cognition and their dysfunction underlies diverse diseases. In both the resting and desensitized states of AMPA and kainate receptor subtypes, the ion channels are closed, whereas the ligand-binding domains, which are physically coupled to the channels, adopt markedly different conformations. Without an atomic model for the desensitized state, it is not possible to address a central problem in receptor gating: how the resting and desensitized receptor states both display closed ion channels, although they have major differences in the quaternary structure of the ligand-binding domain. Here, by determining the structure of the kainate receptor GluK2 subtype in its desensitized state by cryo-electron microscopy (cryo-EM) at 3.8 Å resolution, we show that desensitization is characterized by the establishment of a ring-like structure in the ligand-binding domain layer of the receptor. Formation of this 'desensitization ring' is mediated by staggered helix contacts between adjacent subunits, which leads to a pseudo-four-fold symmetric arrangement of the ligand-binding domains, illustrating subtle changes in symmetry that are important for the gating mechanism. Disruption of the desensitization ring is probably the key switch that enables restoration of the receptor to its resting state, thereby completing the gating cycle.

Structural mechanism of glutamate receptor activation and desensitization.

Ionotropic glutamate receptors are ligand-gated ion channels that mediate excitatory synaptic transmission in the vertebrate brain. To gain a better understanding of how structural changes gate ion flux across the membrane, we trapped rat AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) and kainate receptor subtypes in their major functional states and analysed the resulting structures using cryo-electron microscopy. We show that transition to the active state involves a 'corkscrew' motion of the receptor assembly, driven by closure of the ligand-binding domain. Desensitization is accompanied by disruption of the amino-terminal domain tetramer in AMPA, but not kainate, receptors with a two-fold to four-fold symmetry transition in the ligand-binding domains in both subtypes. The 7.6 Å structure of a desensitized kainate receptor shows how these changes accommodate channel closing. These findings integrate previous physiological, biochemical and structural analyses of glutamate receptors and provide a molecular explanation for key steps in receptor gating.

Ion-dependent gating of kainate receptors.

Ligand-gated ion channels are an important class of signalling protein that depend on small chemical neurotransmitters such as acetylcholine, l-glutamate, glycine and gamma-aminobutyrate for activation. Although numerous in number, neurotransmitter substances have always been thought to drive the receptor complex into the open state in much the same way and not rely substantially on other factors. However, recent work on kainate-type (KAR) ionotropic glutamate receptors (iGluRs) has identified an exception to this rule. Here, the activation process fails to occur unless external monovalent anions and cations are present. This absolute requirement of ions singles out KARs from all other ligand-gated ion channels, including closely related AMPA- and NMDA-type iGluR family members. The uniqueness of ion-dependent gating has earmarked this feature of KARs as a putative target for the development of selective ligands; a prospect all the more compelling with the recent elucidation of distinct anion and cation binding pockets. Despite these advances, much remains to be resolved. For example, it is still not clear how ion effects on KARs impacts glutamatergic transmission. I conclude by speculating that further analysis of ion-dependent gating may provide clues into how functionally diverse iGluRs families emerged by evolution. Consequently, ion-dependent gating of KARs looks set to continue to be a subject of topical inquiry well into the future.

Kainate receptors are primarily postsynaptic to SP-containing axon terminals in the trigeminal dorsal horn.

Kainate receptors (KARs) are involved in the modulation and transmission of nociceptive information from peripheral afferents to neurons in the spinal cord and trigeminal dorsal horns. KARs are found at both pre- and postsynaptic sites in the dorsal horn. We hypothesized that KARs and Substance P (SP), a modulatory neuropeptide that is used as a marker of nociceptive afferents, have a complex interactive relationship. To determine the cellular relationship and connectivity between KARs and SP afferents, we used electron microscopic dual immunocytochemical analysis to examine the ultrastructural localization of KAR subunits GluR5, 6 and 7 (GluR5,6,7) in relation to SP within laminae I and II in the rat trigeminal dorsal horn. KARs were distributed both postsynaptically in dendrites and somata (51% of GluR5,6,7 immunoreactive (-ir) profiles) and presynaptically in axons and axon terminals (45%). We also found GluR5,6,7-ir glial profiles (5%). The majority of SP-ir profiles were presynaptic axons and axon terminals. SP-ir dendritic profiles were rare, yet 23% contained GluR5,6,7 immunoreactivity. GluR5,6,7 and SP were also colocalized at presynaptic sites (18% of GluR5,6,7-ir axons and axon terminals contained SP; while 11% of SP-ir axons and axon terminals contained GluR5,6,7). The most common interaction between KARs and SP we observed was GluR5,6,7-ir dendrites contacted by SP-ir axon terminals; 54% of the dendritic targets of SP-ir axon terminals were GluR5,6,7-ir. These results provide anatomical evidence that KARs primarily mediate nociceptive transmission postsynaptic to SP-containing afferents and may also modulate the presynaptic release of SP and glutamate in trigeminal dorsal horn.

Subcellular localization and trafficking of kainate receptors.

Glutamate receptors of the kainate type have been identified recently as key players in the modulation of neuronal-network activity. The role of kainate receptors depends on their precise subcellular localization in presynaptic, postsynaptic and extrasynaptic domains. Subcellular localization of kainate receptors has been inferred mainly from electrophysiological studies with the help of selective pharmacological tools and kainate receptor mutant mice. These studies, combined with recent ultrastructural data, highlight the diversity of subcellular localizations of kainate receptors. It is important to understand the molecular mechanisms that underlie the polarized trafficking of kainate receptors in distinct neuronal domains. In this article, we review recent data that shed light on the trafficking and membrane delivery of kainate receptor isoforms, and on the identification of proteins that interact with kainate receptors and might regulate this trafficking.

Pre-synaptic kainate receptors in GABAergic and glutamatergic axon terminals in the monkey globus pallidus.

Although the localization and role of kainate receptors in the CNS remain poorly known, complex, and rather unusual, pre-synaptic auto- and heteroreceptor functions have been disclosed in various brain regions. Basal ganglia nuclei, including the globus pallidus, are enriched in GluR6/7 immunoreactivity. Using electron microscopic immunocytochemistry for GluR6/7 combined with post-embedding immunogold labeling for GABA, we demonstrate that GluR6/7 immunoreactivity is enriched in a large subpopulation of small unmyelinated, presumably pre-terminal, axons as well as GABAergic and putative glutamatergic axon terminals in the internal and external segments of the globus pallidus in monkey. Our findings suggest that kainate receptors are located to subserve pre-synaptic modulation of inhibitory and excitatory transmission in the primate globus pallidus.

High level of mGluR7 in the presynaptic active zones of select populations of GABAergic terminals innervating interneurons in the rat hippocampus.

The release of neurotransmitters is modulated by presynaptic metabotropic glutamate receptors (mGluRs), which show a highly selective expression and subcellular location in glutamatergic terminals in the hippocampus. Using immunocytochemistry, we investigated whether one of the receptors, mGluR7, whose level of expression is governed by the postsynaptic target, was present in GABAergic terminals and whether such terminals targeted particular cells. A total of 165 interneuron dendritic profiles receiving 466 synapses (82% mGluR7a-positive) were analysed. The presynaptic active zones of most GAD-(77%) or GABA-positive (94%) synaptic boutons on interneurons innervated by mGluR7a-enriched glutamatergic terminals (mGluR7a-decorated) were immunopositive for mGluR7a. GABAergic terminals on pyramidal cells and most other interneurons in str. oriens were mGluR7a-immunonegative. The mGluR7a-decorated cells were mostly somatostatin- and mGluR1alpha-immunopositive neurons in str. oriens and the alveus. Their GABAergic input mainly originated from VIP-positive terminals, 90% of which expressed high levels of mGluR7a in the presynaptic active zone. Parvalbumin-positive synaptic terminals were rare on mGluR7a-decorated cells, but on these neurons 73% of them were mGluR7a-immunopositive. Some type II synapses innervating interneurons were immunopositive for mGluR7b, as were some type I synapses. Because not all target cells of VIP-positive neurons are known it has not been possible to determine whether mGluR7 is expressed in a target-cell-specific manner in the terminals of single GABAergic cells. The activation of mGluR7 may decrease GABA release to mGluR7-decorated cells at times of high pyramidal cell activity, which elevates extracellular glutamate levels. Alternatively, the presynaptic receptor may be activated by as yet unidentified endogenous ligands released by the GABAergic terminals or the postsynaptic dendrites.

Development of glutamatergic synapses in the rat retina: the postnatal expression of ionotropic glutamate receptor subunits.

We examined the distribution of the AMPA glutamate receptor subunits GluR1 to GluR4, of the kainate receptor subunits GluR6/7 and KA2, and of the glutamate receptor subunits delta1/2, during postnatal development of the rat retina by immunocytochemistry and light microscopy using receptor subunit specific antisera. The various ionotropic glutamate receptor subunits were expressed early in postnatal rat retina, and most of the subunits, with the exception of delta1/2. were found in both synaptic layers of rat retina. The glutamate receptor subunits studied showed differences in their time of appearance, their spatial distribution patterns, and in their expression levels in the developing rat retina. Interestingly, most of the AMPA receptor subunits were expressed earlier than the kainate receptor subunits in the two synaptic layers of the retina, indicating that AMPA glutamate receptors play an important role in early postnatal glutamatergic synaptic transmission. We also studied the ultrastructural localization of the AMPA glutamate receptor subunits GluR1 to GluR4 by immunocytochemistry and electron microscopy in the inner plexiform layer of the mature rat retina. Most of the subunits were found postsynaptic to the ribbon synapses of OFF-cone, ON-cone, and rod bipolar cells. The results of this study suggest an involvement of ionotropic glutamate receptors in processes of synaptic maturation and the formation of synaptic circuitries in the developing plexiform layers of the retina. Furthermore, AMPA and kainate receptors play a role in synaptic processing and in the development of both the scotopic and photopic pathways in the rat retina.

Synaptic distribution of ionotropic glutamate receptors in the inner plexiform layer of the primate retina.

The distribution and synaptic clustering of glutamate receptors (GluRs) were studied in the inner plexiform layer (IPL) of the macaque monkey retina by using subunit specific antisera. A punctate immunofluorescence pattern was observed in the IPL for all subunits tested, and electron microscopy confirmed that the immunoreactive puncta represent clustering of receptors at sites postsynaptic to the bipolar cell ribbon synapses (dyads). Usually only one of the two postsynaptic processes at the dyads expressed a given subunit. Immunoreactive GluR2, GluR2/3, and GluR4 puncta were found at high density throughout the IPL and are probably expressed at every dyad. The GluR1 subunit was expressed at lower density. The N-methyl-D-aspartate (NMDA) receptor subunits NR2A and NR1C2' were restricted to synapses localized in two broad bands in the center of the IPL. They were often colocalized with GluR2/3 and GluR4 subunits. The orphan receptor subunits delta 1/2 predominated in three horizontal bands. The kainate receptor subunits GluR6/7 were clustered in large postsynaptic densities adjacent to bipolar cell axon terminals but lacking a synaptic ribbon on the presynaptic side. This might represent a conventional synapse made by a bipolar axon terminal. The results suggest that GluR2/3 and GluR4, together with NMDA receptors, are preferentially expressed on ganglion cell dendrites, whereas kainate receptors and the delta 1/2 subunits are mostly localized on amacrine cell processes.

Selective synaptic distribution of AMPA and kainate receptor subunits in the outer plexiform layer of the carp retina.

The subunit composition of ionotropic glutamate receptors (GluRs) is extremely diverse and responsible for the diversity of postsynaptic responses to the release of glutamate, which is the major excitatory neurotransmitter in the retina. To understand the functional consequences of this diversity, it is necessary to reveal the synaptic localization and subunit composition of GluRs. We have used immuno light and electron microscopy to localize AMPA and kainate (GluR1, GluR2/3, GluR4, GluR5-7) subunits in identified carp retinal neurons contributing to the outer plexiform layer. GluR1 could not be detected within the outer plexiform layer. Rod and cone horizontal cells all express only GluR2/3 at the tips of their invaginating dendrites. These receptors are also inserted into the membrane of spinules, light-dependent protrusions of the horizontal cell dendrites, flanking the synaptic ribbon of the cone synapse. Bipolar cells express GluR2/3, GluR4, and GluR5-7 at their terminal dendrites invaginating cone pedicles and rod spherules. Colocalization data suggest that each subunit is expressed by a distinct bipolar cell type. The majority of bipolar cells expressing these receptors seem to be of the functional OFF-type; however, in a few instances, GluR2/3 could also be detected on dendrites of bipolar cells that, based on their localization within the cone synaptic complex, appeared to be of the functional ON-type. The spatial arrangement of the different subunits within the cavity of the cone pedicle appeared not to be random: GluR2/3 was found predominantly at the apex of the cavity, GluR4 at its base and GluR5-7 dispersed between the two.

Ultrastructural analysis of NMDA, AMPA, and kainate receptors on unmyelinated and myelinated axons in the periphery.

The present study determines the proportions of unmyelinated cutaneous axons at the dermal-epidermal junction in glabrous skin and of myelinated and unmyelinated axons in the sural and medial plantar nerves that immunostain for subunits of the ionotropic glutamate receptors. Approximately 20% of the unmyelinated cutaneous axon profiles at the dermal-epidermal junction immunostain for either N-methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), or kainate receptor subunits. These findings are consistent with previous observations that NMDA and non-NMDA antagonists ameliorate nociceptive behaviors that result from noxious peripheral stimulation. In the sural nerve, where the large majority of myelinated fibers are sensory, approximately half of the myelinated axon profiles immunostain for the NMDA receptor 1 (R1) subunit, 28% immunostain for the glutamate receptor 1 (GluR1) AMPA subunit, and 11% for the GluR5,6,7 kainate subunits. Even higher proportions immunostain for these receptors in the medial plantar nerve, a mixed sensory and motor nerve. In the sural nerve, 20% of the unmyelinated axon profiles immunostain for NMDAR1 and only 7% label for GluR1 or GluR5,6,7. Because the sural nerve innervates hairy skin, these data suggest that glutamate will activate a higher proportion of unmyelinated axons in glabrous skin than in hairy skin. Measurements of fiber diameters indicate that all sizes of myelinated axon profiles, including Adelta and Abeta, are positively labeled for the ionotropic receptors. The presence of glutamate receptors on large-diameter myelinated axons suggests that these mechanosensitive receptors, presumably transducing touch and pressure, may also respond to local glutamate and thus be chemosensitive.

Effects of propofol on various AMPA-, kainate- and NMDA-selective glutamate receptor channels expressed in Xenopus oocytes.

Effects of a general intravenous anesthetic 2,6-diisopropylphenol (propofol) on various glutamate receptor (GluR) channels were examined on the alpha 1 and alpha 1/alpha 2 GluR channels selective for alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA), the beta 2/gamma 2 GluR channels selective for kainate, and the epsilon 2/zeta 1 and epsilon 3/zeta 1 N-methyl-D-aspartate (NMDA) receptor channels expressed in Xenopus oocytes. Propofol suppressed the current responses of the alpha 1/alpha 2, beta 2/gamma 2, epsilon 2/zeta 1 and epsilon 3/zeta 1 channels in a dose-dependent manner, whereas it enhanced the current responses of the alpha 1 channel. The extents of inhibition were in the order epsilon 2/zeta 1 > epsilon 3/zeta 1 > beta 2/gamma 2 > alpha 1/alpha 2 channels. During perfusion of 500 microM propofol, the alpha 1/alpha 2, beta 2/gamma 2, epsilon 2/zeta 1 and epsilon 3/zeta 1 channels were progressively suppressed. Furthermore, 10 min perfusion of 20 microM propofol inhibited the epsilon 2/zeta 1 channel by 24%. These results suggest that clinical concentrations (approximately 35 microM) of propofol suppress the NMDA receptor channels slightly.

A topological analysis of goldfish kainate receptors predicts three transmembrane segments.

Glutamate receptors are the most abundant excitatory neurotransmitter receptors in vertebrate brain. We have previously cloned cDNAs encoding two homologous kainate receptors (GFKAR alpha, 45 kDa, and GFKAR beta, 41 kDa) from goldfish brain and proposed a topology with three transmembrane domains (Wo, Z. G., and Oswald, R. E. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 7154-7158). These studies have been extended using an in vitro translation/translocation system in conjunction with site-specific antibodies and point and deletion mutations. We report here that the entire region between the previously proposed third and fourth transmembrane segments is translocated and likely to be extracellular in mature receptors. This was based on the following results. 1) The entire segment was protected from Proteinase K and trypsin digestion and could be immunoprecipitated by a site-specific antibody. 2) Functional sites for N-glycosylation are present in the C-terminal half of the segment, and 3) a mutation, constructed with an additional consensus site for N-glycosylation in the N-terminal half of the segment, was found to be glycosylated at that site. Given the fact that the N terminus of the protein is likely to be extracellular, this would place an even number of transmembrane segments between the extracellular N terminus and the glycosylated segment. In addition, results of N-glycosylation and proteolysis protection assays of GFKAR alpha mutations indicated that the previously proposed second transmembrane segment is not a true transmembrane domain. These results provide further evidence in support of a topology with three transmembrane domains that has important implications for the relationship of structure to function in ionotropic glutamate receptors.

Cellular and synaptic localization of NMDA and non-NMDA receptor subunits in neocortex: organizational features related to cortical circuitry, function and disease.

Excitatory amino acid (EAA) receptors are an important component of neocortical circuitry as a result of their role as the principal mediators of excitatory synaptic activity, as well as their involvement in use-dependent modifications of synaptic efficacy, excitoxicity and cell death. The diversity in the effects generated by EAA-receptor activation can be attributed to multiple receptor subtypes, each of which is composed of multimeric assemblies of functionally distinct receptor subunits. The use of subunit-specific antibodies and molecular probes now makes it feasible to localize individual receptor subunits anatomically with a high level of cellular and synaptic resolution. Initial studies of the distribution of immunocytochemically localized EAA-receptor subunits suggest that particular subunit combinations exhibit a differential cellular, laminar and regional distribution in the neocortex. While such patterns might indicate that the functional heterogeneity of EAA-receptor-linked circuits, and the cell types in which they operate, are based partly on differential subunit parcellation, a definitive integration of these anatomical details into current schemes of cortical circuitry and organization awaits many further studies. Ideally, such studies should link a high level of molecular precision regarding subunit localization with synaptic details of identified connections and neurochemical features of neocortical cells.