A glutamate receptor channel with high affinity for domoate and kainate.

The non-NMDA family of glutamate receptors comprises a growing number of structurally related subunits (GluR-A to -D or -1 to -4; GluR-5, -6; KA-1). GluR-A to -D appear to constitute the major AMPA receptor subtypes but the functional and pharmacological characteristics of the other subunits are unresolved. Using a mammalian expression system we demonstrate here that homomeric GluR-5 receptors exhibit properties of a high affinity domoate (KD approximately 2 nM) and kainate (KD approximately 70 nM) binding site. For these receptors, the rank order of ligands competing with [3H]kainate binding was domoate much greater than quisqualate approximately glutamate much greater than AMPA approximately CNQX. The respective receptor channels were gated in decreasing order of sensitivity by domoate, kainate, glutamate and AMPA. In contrast to recombinantly expressed GluR-A to -D channels, currents elicited at GluR-5 receptor desensitize channels to all agonists. This property is characteristic of currents in peripheral neurons on sensory ganglia. These findings suggest the existence of at least two distinct types of non-NMDA receptor channels, both gated by AMPA and kainate, but differing in pharmacology and current properties.     

Functional expression from cloned cDNAs of glutamate receptor species responsive to kainate and quisqualate.

The complete amino acid sequences of two mouse glutamate receptor subunits (GluR1 and GluR2) have been deduced by cloning and sequencing the cDNAs. Xenopus oocytes injected with mRNA derived from the GluR1 cDNA exhibit current responses both to kainate and to quisqualate as well as to glutamate, whereas oocytes injected with mRNA derived from the GluR2 cDNA show little response. Injection of oocytes with both the mRNAs produces current responses larger than those induced by the GluR1-specific mRNA and the dose-response relations indicate a positively cooperative interaction between the two subunits. These results suggest that kainate and quisqualate can activate a common glutamate receptor subtype and that glutamate-gated ionic channels are hetero-oligomers of different subunits.     

A chimeric glutamate receptor subunit: discrete changes modify the properties of the channel.

GluR1 and GluR2 are two highly homologous subunits of the glutamate AMPA receptor but with different functional properties. In ligand gated channels the transmembrane domain II is thought to form the wall of the ionic pore and determine the electrical properties. A chimeric AMPA receptor subunit was constructed by replacing the region comprising transmembrane domains I and II in GluR1 by the corresponding region of GluR2. Alone or forming an heteromer with GluR1, the resulting chimera has the properties of GluR2. Sequence comparison suggests that an arginine at position 600 in the chimera instead of a glutamine in GluR1 is responsible for these properties.     

Cloning and expression of the epsilon 4 subunit of the NMDA receptor channel.

The primary structure of a novel subunit of the mouse NMDA (N-methyl-D-aspartate) receptor channel, designated epsilon 4, has been revealed by cloning and sequencing the cDNA. The epsilon 4 subunit shares high amino acid sequence identity with the epsilon 1, epsilon 2 and epsilon 3 subunits of the mouse NMDA receptor channel, thus constituting the epsilon subfamily of the glutamate receptor channel. Expression from cloned cDNAs of the epsilon 4 subunit together with the zeta 1 subunit in Xenopus oocytes yields functional NMDA receptor channels. The epsilon 4/zeta 1 heteromeric channel exhibits high apparent affinities for agonists and low sensitivities to competitive antagonists. The epsilon 4 subunit is thus distinct in functional properties from the epsilon 1, epsilon 2 and epsilon 3 subunits, and contributes further diversity of the NMDA receptor channel.     

Emerging models of glutamate receptor ion channel structure and function

Excitatory synaptic transmission in the brain is mediated by ligand-gated ion channels (iGluRs) activated by glutamate. Distinct from other neurotransmitter receptors, the extracellular domains of iGluRs are loosely packed assemblies with two clearly distinct layers, each of which has both local and global 2-fold axes of symmetry. By contrast, the iGluR transmembrane segments have 4-fold symmetry and share a conserved pore loop architecture found in tetrameric voltage-gated ion channels. The striking layered architecture of iGluRs revealed by the 3.6?? resolution structure of an AMPA receptor homotetramer likely arose from gene fusion events that occurred early in evolution. Although this modular design has greatly facilitated biophysical and structural studies on individual iGluR domains, and suggested conserved mechanisms for iGluR gating, recent work is beginning to reveal unanticipated diversity in the structure, allosteric regulation, and assembly of iGluR subtypes.     

Primary structure and functional expression from cDNA of the cardiac ryanodine receptor/calcium release channel

The sequence of 4968 (or 4976 with an insertion) amino acids composing the ryanodine receptor from rabbit cardiac sarcoplasmic reticulum has been deduced by cloning and sequencing the cDNA. This protein is homologous in amino acid sequence and shares characteristic structural features with the skeletal muscle ryanodine receptor. Xenopus oocytes injected with mRNA derived from the cardiac ryanodine receptor cDNA exhibit Ca2(+)-dependent Cl- current in response to caffeine, which indicates the formation of functional calcium release channels. RNA blot hybridization analysis with a probe specific for the cardiac ryanodine receptor mRNA shows that the stomach and brain contain a hybridizable RNA species with a size similar to that of the cardiac mRNA. This result, in conjunction with cloning and analysis of partial cDNA sequences, suggests that the brain contains a cardiac type of ryanodine receptor mRNA.     

Evidence for a single glutamate receptor of the ionotropic kainate/quisqualate type

The kainate (KA) and the quisqualate (QUIS) receptors that activate cation channels in the central nervous system have previously been defined as two of the major glutamate receptor types. In amphibian brain, an exceptionally rich source of these sites, they can be coextracted by octylglucoside and shown to behave as one entity in all analyses made in solution. When partly purified by lectin affinity, ion-exchange chromatography or by sucrose gradient centrifugation, the two activities comigrate in a 1:1 ratio. When the QUIS component is bound to an immobilized specific QUIS agonist, the KA component is extracted in parallel with it. There are equivalent numbers of the QUIS and KA sites and the two sites show a single affinity series for the binding of glutamatergic agonists. We deduce that KA or QUIS select different conformations of a single KA/QUIS receptor binding site, leading thus to the different channel-opening events that have been reported for these two agonists.     

Pyrimidine methyl anilines: selective potentiators for the metabotropic glutamate 2 receptor

Pyrimidine methyl anilines as potent and selective mGlu2 potentiators are described. Findings from the structure-activity-relationship investigations are discussed.     

A cluster of three novel Ca2+ channel gamma subunit genes on chromosome 19q13.4: evolution and expression profile of the gamma subunit gene family

The CACNG1 gene on chromosome 17q24 encodes an integral membrane protein that was originally isolated as the regulatory gamma subunit of voltage-dependent Ca2+ channels from skeletal muscle. The existence of an extended family of gamma subunits was subsequently demonstrated upon identification of CACNG2 (22q13), CACNG3 (16p12-p13), and CACNG4 and CACNG5 (17q24). In this study, we describe a cluster of three novel gamma subunit genes, CACNG6, CACNG7, and CACNG8, located in a tandem array on 19q13.4. Phylogenetic analysis indicates that this array is paralogous to the cluster containing CACNG1, CACNG5, and CACNG4, respectively, on chromosome 17q24. We developed sensitive RT-PCR assays and examined the expression profile of each member of the gamma subunit gene family, CACNG1-CACNG8. Analysis of 24 human tissues plus 3 dissected brain regions revealed that CACNG1 through CACNG8 are all coexpressed in fetal and adult brain and differentially transcribed among a wide variety of other tissues. The expression of distinct complements of gamma subunit isoforms in different cell types may be an important mechanism for regulating Ca2+ channel function.     

Single channel kinetics of a glutamate receptor.

The glutamate receptor-channel of locust muscle membrane was studied using the patch-clamp technique. Muscles were pretreated with concanavalin A to block receptor-channel desensitization, thus facilitating analysis of receptor-channel gating kinetics. Single channel kinetics were analyzed to aid in identification of the molecular basis of channel gating. Channel dwell-time distributions and dwell-time autocorrelation functions were calculated from single channel data recorded in the precence of 10-4M glutamate. Analysis of the dwell time distributions in terms of mixtures of exponential functions revealed there to be at least three open states of the receptor-channel and at least four closed states. Autocorrelation function analysis showed there to be at least three pathways linking the open states with the closed. This results in a minimal scheme for gating of the glutamate receptor-channel, which is suggestive of allosteric models of receptor-channel gating.     

SUMOylation of the kainate receptor subunit GluK2 contributes to the activation of the MLK3-JNK3 pathway following kainate stimulation

Protein SUMOylation has been implicated in the pathogenesis of ischemic stroke. However, the underlying mechanisms remain unclear. Here, we found that global brain ischemia evokes a sustained elevation of GluK2 SUMOylation in the rat hippocampal CA1 region. Over-expression of wild-type GluK2, but not SUMOylation-deficient mutant, significantly increased the activity of MLK3 and JNK3 after kainate stimulation. SUMOylation deficiency attenuated the kainate-stimulated interaction between MLK3 and GluK2. In addition, inhibition of kainate-evoked GluK2 endocytosis decreased the activation of MLK3-JNK3 signaling and the binding of MLK3-GluK2 in cultured cortical neurons. These results suggest that the internalization of GluK2 following SUMO modification promotes its binding with MLK3, thereby activating the MLK3-JNK3 pathway, which may be responsible for ischemic neuronal cell death.     

RNA aptamers selected against the GluR2 glutamate receptor channel

The excessive activation of AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors, a subtype of glutamate ion channels, has been implicated in various neurological diseases such as cerebral ischemeia and amyotrophic lateral sclerosis. Inhibitors of AMPA receptors are drug candidates for potential treatment of these diseases. Using the systematic evolution of ligands by exponential enrichment (SELEX), we have selected a group of RNA aptamers against the recombinant GluR2Qflip AMPA receptor transiently expressed in HEK-293 (human embryonic kidney) cells. One of the aptamers, AN58, is shown to competitively inhibit the receptor. The nanomolar affinity of AN58 rivals that of NBQX (6-nitro-7-sulfamoyl-benzo[f]quinoxaline-2,3-dione), one of the best competitive inhibitors. Like NBQX, AN58 has the highest affinity for GluR2, the selection target, among all AMPA receptor subunits. However, AN58 has a higher selectivity for the GluR4 AMPA receptor subunit and remains potent even at pH = 6.8 (i.e., a clinically relevant acidic pH), as compared with NBQX. Furthermore, this RNA molecule possesses stable physical properties. Therefore, AN58 serves as a unique lead compound for developing water-soluble inhibitors with a nanomolar affinity for GluR2 AMPA receptors.     

Cloning and characterization of a GABAA receptor gamma2 subunit variant

We have cloned a novel gamma-aminobutyric acid type A (GABAA) receptor gamma2 subunit variant named gamma2XL. gamma2XL contains an alternatively spliced exon, resulting in the addition of 40 amino acids to the N-terminal extracellular domain between Ser171 and Tyr172. We show that gamma2XL failed to localize to the cell surface when it was coexpressed with the alpha2 and beta1 subunits in human embryonic kidney 293 cells. Expression of gamma2XL in 293 cells suppressed GABAA receptor binding in a dose-dependent manner by preventing GABAA receptor cell-surface localization. We also generated a gamma2 mutant with Ser171 and Tyr172 converted to glycine and threonine, respectively. We demonstrate that this mutant has a significantly lower affinity for the alpha2 and beta1 subunits and failed to reach the cell surface when coexpressed with these subunits. Together, our results indicate that Ser171 and Tyr172 in the gamma2 subunit constitute a critical motif. When this motif is disrupted by insertion of the alternative exon, access of the gamma2 subunit to the cell surface is prevented.