GluK2 Kainate Research Articles

Cochlear Kainate Receptors

Synaptic transmission between the cochlear hair cell and its afferent fiber is mediated by glutamate receptors. While kainate receptors are known to be present in the spiral ganglion, little is known of their distribution or functional role. We have detected all five kainate receptor subunits in the mouse cochlea with quantitative RT-PCR and with immunohistochemistry. We observed kainate receptors on afferent terminals co-localized with α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA: ) receptors at the afferent synapse. Individual terminals innervating a single hair cell varied in their ratios of AMPA: to kainate receptor immunoreactivity. Infusion of the mouse cochlea via the scala tympani with UBP296, a recently developed antagonist with high specificity for the GluK1 kainate receptor (compared to the AMPA: receptor), reduced the compound action potential and elevated auditory neural thresholds without affecting the distortion product otoacoustic emission thresholds. Thus, the pharmacological evidence suggests that kainate receptors may contribute to the response to transmitter released from the hair cell during acoustic stimulation. It is plausible that afferent transmission at this synapse is mediated by a mix of AMPA: and kainate receptors.

Binding and Selectivity of the Marine Toxin Neodysiherbaine A and Its Synthetic Analogues to GluK1 and GluK2 Kainate Receptors

Dysiherbaine (DH) and neodysiherbaine A (NDH) selectively bind and activate two kainate-type ionotropic glutamate receptors, GluK1 and GluK2. The ligand-binding domains of human GluK1 and GluK2 were crystallized as bound forms with a series of DH analogues including DH, NDH, 8-deoxy-NDH, 9-deoxy-NDH and 8,9-dideoxy-NDH (MSVIII-19), isolated from natural sources or prepared by total synthesis. Since the DH analogues exhibit a wide range of binding affinities and agonist efficacies, it follows that the detailed analysis of crystal structure would provide us with a significant opportunity to elucidate structural factors responsible for selective binding and some aspects of gating efficacy. We found that differences in three amino acids (Thr503, Ser706 and Ser726 in GluK1 and Ala487, Asn690 and Thr710 in GluK2) in the ligand-binding pocket generate differences in the binding modes of NDH to GluK1 and GluK2. Furthermore, deletion of the C9 hydroxy group in NDH alters the ligand conformation such that it is no longer suited for binding to the GluK1 ligand-binding pocket. In GluK2, NDH pushes and rotates the side chain of Asn690 (substituted for Ser706 in GluK1) and disrupts an interdomain hydrogen bond with Glu409. The present data support the idea that receptor selectivities of DH analogues resulted from the differences in the binding modes of the ligands in GluK1/GluK2 and the steric repulsion of Asn690 in GluK2. All ligands, regardless of agonist efficacy, induced full domain closure. Consequently, ligand efficacy and domain closure did not directly coincide with DH analogues and the kainate receptors.

Contributions of area Te2 to rat recognition memory.

Ablations and local intracerebral infusions were used to determine the role of rat temporal association cortex (area Te2) in object recognition memory, so that this role might be compared with that of the adjacent perirhinal cortex (PRH). Bilateral lesions of Te2 impaired recognition memory measured by preferential exploration of a novel rather than a familiar object at delays ≥20 min but not after a 5-min delay. Local infusion bilaterally into Te2 of (1) CNQX to block AMPA/kainate receptors or (2) lidocaine to block axonal transmission or (3) AP5, an NMDA receptor antagonist, impaired recognition memory after a 24-h but not a 20-min delay. In PRH all these manipulations impair recognition memory after a 20-min as well as a 24-h delay. UBP302, a GluK1 kainate receptor antagonist, impaired recognition memory after a 24-h but not a 20-min delay, contrasting with its action in PRH where it impairs only shorter-term (20 min) recognition memory. Also in contrast to PRH, infusion of the muscarinic receptor antagonist scopolamine was without effect. The Te2 impairments could not readily be ascribed to perceptual deficits. Hence, Te2 is essential for object recognition memory at delays >5 or 20 min. Thus, at long delays both area Te2 and PRH are necessary for object recognition memory.

Synaptic Targeting and Functional Modulation of GluK1 Kainate Receptors by the Auxiliary Neuropilin and Tolloid-Like (NETO) Proteins

Auxiliary proteins modify the biophysical function and pharmacological properties of ionotropic glutamate receptors and likely are important components of receptor signaling complexes in vivo. The neuropilin and tolloid-like proteins (NETO) 1 and NETO2, two closely related CUB domain-containing integral membrane proteins, were identified recently as auxiliary proteins that slowed GluK2a kainate receptor current kinetics without impacting receptor membrane localization. Here we demonstrate that NETO2 profoundly slows the desensitization rate of GluK1 kainate receptors, promotes plasma membrane localization of transfected receptors in heterologous cells and rat hippocampal neurons, and targets GluK1-containing receptors to synapses. Conversely, the closely related protein NETO1 increases the rate of GluK1 receptor desensitization. Incorporation of NETO proteins into kainate receptor-signaling complexes therefore extends the temporal range of receptor gating by over an order of magnitude. The presence of these auxiliary proteins could underlie some of the unusual aspects of kainate receptor function in the mammalian CNS.

Antinociceptive effects of MSVIII-19, a functional antagonist of the GluK1 kainate receptor

The ionotropic glutamate receptor subunit, GluK1 (GluR5), is expressed in many regions of the nervous system related to sensory transmission. Recently, a selective ligand for the GluK1 receptor, MSVIII-19 (8,9-dideoxy-neodysiherbaine), was synthesized as a derivative of dysiherbaine, a toxin isolated from the marine sponge Lendenfeldia chondrodes. MSVIII-19 potently desensitizes GluK1 receptors without channel activation, rendering it useful as a functional antagonist. Given the high selectivity for GluK1 and the proposed role for this glutamate receptor in nociception, we sought to test the analgesic potential of MSVIII-19 in a series of models of inflammatory, neuropathic, and visceral pain in mice. MSVIII-19 delivered intrathecally dose-dependently reduced formalin-induced spontaneous behaviors and reduced thermal hypersensitivity 3 hours after formalin injection and 24 hours after complete Freund’s adjuvant-induced inflammation, but had no effect on mechanical sensitivity in the same models. Intrathecal MSVIII-19 significantly reduced both thermal hyperalgesia and mechanical hypersensitivity in the chronic constriction injury model of neuropathic pain, but had no effect in the acetic acid model of visceral pain. Peripheral administration of MSVIII-19 had no analgesic efficacy in any of these models. Finally, intrathecal MSVIII-19 did not alter responses in Tail-flick tests or performance on the accelerating RotaRod. These data suggest that spinal administration of MSVIII-19 reverses hypersensitivity in several models of pain in mice, supporting the clinical potential of GluK1 antagonists for the management of pain. Spinal administration of a GluK1 antagonist shows analgesic efficacy in several mouse models, supporting the clinical potential of GluK1 antagonists for pain management.

Exploring kainate receptor pharmacology using molecular dynamics simulations

Ionotropic glutamate receptors (iGluRs) are enticing targets for pharmaceutical research; however, the search for selective ligands is a laborious experimental process. Here we introduce a purely computational procedure as an approach to evaluate ligand-iGluR pharmacology. The ligands are docked into the closed ligand-binding domain and during the molecular dynamics (MD) simulation the bi-lobed interface either opens (partial agonist/antagonist) or stays closed (agonist) according to the properties of the ligand. The procedure is tested with closely related set of analogs of the marine toxin dysiherbaine bound to GluK1 kainate receptor. The modeling is set against the abundant binding data and electrophysiological analyses to test reproducibility and predictive value of the procedure. The MD simulations produce detailed binding modes for analogs, which in turn are used to define structure-activity relationships. The simulations suggest correctly that majority of the analogs induce full domain closure (agonists) but also distinguish exceptions generated by partial agonists and antagonists. Moreover, we report ligand-induced opening of the GluK1 ligand-binding domain in free MD simulations. The strong correlation between in silico analysis and the experimental data imply that MD simulations can be utilized as a predictive tool for iGluR pharmacology and functional classification of ligands.

A Role for SNAP25 in Internalization of Kainate Receptors and Synaptic Plasticity

Regulation of surface insertion and internalization of AMPA and NMDA receptors has emerged as a key mechanism for the control of synaptic strength. Regulatory elements for synaptic kainate receptors (KARs) are, however, largely undetermined. We have found that SNAP25 is critical for the synaptic removal of KARs, acting via GluK5 (i.e., KA2) subunits. SNAP25 coimmunoprecipitates with protein complexes containing PICK1, GRIP1, and GluK5 and colocalizes with GluK5 in both hippocampal neurons and transfected HEK293 cells. In hippocampal slices, purified SNAP25 antibodies and blocking peptides caused a GluK5-dependent run-up of KARs-mediated EPSC (EPSC(KAR)) recorded from CA3 pyramidal neurons when included in the patch pipette and prevented activity-dependent long-term depression of EPSC(KAR). As EPSC(KAR) LTD, SNAP25/PICK1/GluK5 interactions are dynamically regulated by PKC.

Topiramate Reduces Excitability in the Basolateral Amygdala by Selectively Inhibiting GluK1 (GluR5) Kainate Receptors on Interneurons and Positively Modulating GABAAReceptors on Principal Neurons

Topiramate [2,3:4,5-bis-O-(1-methylethylidene)-beta-D-fructopyranose sulfamate] is a structurally novel antiepileptic drug that has broad efficacy in epilepsy, but the mechanisms underlying its therapeutic activity are not fully understood. We have found that topiramate selectively inhibits GluK1 (GluR5) kainate receptor-mediated excitatory postsynaptic responses in rat basolateral amygdala (BLA) principal neurons and protects against seizures induced by the GluK1 kainate receptor agonist (R,S)-2-amino-3-(3-hydroxy-5-tert-butylisoxazol-4-yl)propanoic acid (ATPA). Here, we demonstrate that topiramate also modulates inhibitory function in the BLA. Using whole-cell recordings in rat amygdala slices, we found that 0.3 to 10 microM topiramate 1) inhibited ATPA-evoked postsynaptic currents recorded from BLA interneurons; 2) suppressed ATPA-induced enhancement of spontaneous inhibitory postsynaptic currents (IPSCs) recorded from BLA pyramidal cells; and 3) blocked ATPA-induced suppression of evoked IPSCs, which is mediated by presynaptic GluK1 kainate receptors present on BLA interneurons. Topiramate (10 microM) had no effect on the AMPA [(R,S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid]-induced enhancement of spontaneous activity of BLA neurons. Thus, although topiramate inhibits GluK1 kainate receptor-mediated enhancement of interneuron firing, it promotes evoked GABA release, leading to a net inhibition of circuit excitability. In addition, we found that topiramate (0.3-10 microM) increased the amplitude of evoked, spontaneous, and miniature IPSCs in BLA pyramidal neurons, indicating an enhancement of postsynaptic GABA(A) receptor responses. Taken together with our previous findings, we conclude that topiramate protects against hyperexcitability in the BLA by suppressing the GluK1 kainate receptor-mediated excitation of principal neurons by glutamatergic afferents, blocking the suppression of GABA release from interneurons mediated by presynaptic GluK1 kainate receptors and directly enhancing GABA(A) receptor-mediated inhibitory currents.

A study of GluK1 kainate receptor polymorphisms in Down syndrome reveals allelic non-disjunction at 1173(C/T).

Mechanisms underlying Down syndrome (DS)-related mental retardation (MR) remain poorly understood. In trisomic offspring, non-disjunction may result in the reduction to homozygosity of a susceptibility allele inherited from a heterozygous parent. Accordingly, we sought evidence for allelic non-disjunction in the GluK1 gene that encodes the critical kainite-binding glutamate receptor subunit-5, maps to chromosome 21q22.1 in the DS critical region and is expressed in brain regions responsible for learning and memory. Three polymorphisms of GluK1 [522(A/C) rs363538; 1173(C/T) rs363430 and 2705(T/C) rs363504] were genotyped in 86 DS patient families by means of PCR-coupled RFLP assays and evaluated with respect to allele frequency, heterozygosity, linkage disequilibrium, stage and parental origin of allelic non-disjunction. We report that the distribution of allele frequencies is in Hardy-Weinberg equilibrium. Moderate heterozygosity (0.339) and a major allele frequency of 0.78 render the 1173(C/T) marker informative. Pair-wise comparisons reveal that 522(A/C)-1173(C/T) [χ2 = 31.2, df = 1, p = 0.0001; D’ = 0.42] and 1173(C/T)-2705(T/C) [χ2 = 18.3, df = 1, p = 0.0001; D’ = 0.34] are in significant linkage disequilibrium of weak magnitude. The estimated ratio of meiosis-I to meiosis-II errors arising from allelic non-disjunction of 1173(C/T) is 4:1 in maternal cases and 2:1 in paternal cases. Studies including additional markers and patient samples are warranted to further substantiate present findings.