Ionotropic glutamate receptors

In the brain, most fast excitatory synaptic transmission is mediated through L-glutamate acting on postsynaptic ionotropic glutamate receptors. These receptors are of two kinds—the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)/kainate (non-NMDA) and theN-methyl-D-aspartate (NMDA) receptors, which are thought to be colocalized onto the same postsynaptic elements. This excitatory transmission can be modulated both upward and downward, long-term potentiation (LTP) and long-term depression (LTD), respectively. Whether the expression of LTP/LTD is pre-or postsynaptically located (or both) remains an enigma. This article will focus on what postsynaptic modifications of the ionotropic glutamate receptors may possibly underly long-term potentiation/depression. It will discuss the character of LTP/LTD with respect to the temporal characteristics and to the type of changes that appears in the non-NMDA and NMDA receptor-mediated synaptic currents, and what constraints these findings put on the possible expression mechanism(s) for LTP/LTD. It will be submitted that if a modification of the glutamate receptors does underly LTP/LTD, an increase/decrease in the number of functional receptors is the most plausible alternative. This change in receptor number will have to include a coordinated change of both the non-NMDA and the NMDA receptors.     

Ionotropic glutamate receptors.

The glutamate-binding sites of ionotropic glutamate receptors are formed from two extracellular domains of a single subunit. Conformational changes induced by agonist binding produce mechanical processes that are translated into ion gating and receptor desensitization. The interactions between macromolecular assemblies of synaptic proteins and ionotropic glutamate receptors, and their subsequent roles in receptor clustering and specificity are being elucidated. Kainate receptor pharmacology is finally revealing its secrets as a result of the availability of selective pharmacological agents.     

Memory in Caenorhabditis elegans is mediated by NMDA-type ionotropic glutamate receptors

Learning and memory are essential processes of both vertebrate and invertebrate nervous systems that allow animals to survive and reproduce. The neurotransmitter glutamate signals via ionotropic glutamate receptors (iGluRs) that have been linked to learning and memory formation; however, the signaling pathways that contribute to these behaviors are still not well understood. We therefore undertook a genetic and electrophysiological analysis of learning and memory in the nematode Caenorhabditis elegans. Here, we show that two genes, nmr-1 and nmr-2, are predicted to encode the subunits of an NMDA-type (NMDAR) iGluR that is necessary for memory retention in C. elegans. We cloned nmr-2, generated a deletion mutation in the gene, and showed that like nmr-1, nmr-2 is required for in vivo NMDA-gated currents. Using an associative-learning paradigm that pairs starvation with the attractant NaCl, we also showed that the memory of a learned avoidance response is dependent on NMR-1 and NMR-2 and that expression of NMDARs in a single pair of interneurons is sufficient for normal memory. Our results provide new insights into the molecular and cellular mechanisms underlying the memory of a learned event.     

Ionotropic glutamate receptors trigger microvesicle-mediated exocytosis of L-glutamate in rat pinealocytes

Rat pinealocytes receive noradrenergic innervation that stimulates melatonin synthesis. Besides melatonin, we showed previously that pinealocytes accumulate L-glutamate in microvesicles and secrete it through an exocytic mechanism. The secreted glutamate binds to the class II metabotropic glutamate receptor and inhibits norepinephrine-stimulated melatonin synthesis in neighboring pinealocytes through an inhibitory cyclic AMP cascade. In this study, it was found that, in addition to metabotropic receptors, pinealocytes express functional ionotropic receptors. RT-PCR and northern analyses indicated the expression of mRNA for GluR1, KA2, and NR2C in pineal gland. The presence of GluR1 protein was confirmed by immunological techniques, but neither KA2 nor NR2C was detected. Consistent with this observation, the presence of (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid or kainate, non-N-methyl-D-aspartate receptor agonists, transiently stimulated increased the intracellular Ca(2+) concentration of cultured pinealocytes, whereas N-methyl-D-aspartate did not. These responses were prevented by 6-cyano-7-nitroquinoxaline-2,3-dione, a selective antagonist for non-N-methyl-D-aspartate receptors, by L-type Ca(2+) channel blockers such as nifedipine, or by omitting Ca(2+) or Na(+) in the medium. In the presence of Ca(2+) and Na(+), (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid or kainate evoked glutamate secretion from the cultured cells, which was prevented by 6-cyano-7-nitroquinoxaline-2,3-dione, L-type Ca(2+) channel blockers, type E or B botulinum neurotoxin, or incubation at <20 degrees C. These results strongly suggest that GluR1 is functionally expressed in pinealocytes and triggers microvesicle-mediated exocytosis of L-glutamate via activation of L-type Ca(2+) channels. It is possible that GluR1 participates in a signaling cascade that enhances and expands the L-glutamate signal throughout the pineal gland.     

Serotonin receptors antagonistically modulate Caenorhabditis elegans longevity

The neurotransmitter serotonin has been implicated in affecting the variation of longevity in natural Drosophila populations and age-related diseases in mammals. Based on these observations, it has been predicted that serotonin signal, perhaps at levels of serotonin biosynthesis, may control lifespan. Here, we investigated a variety of mutations in serotonin-signal genes, including serotonin biosynthesis genes, a serotonin transporter gene, and serotonin receptor genes. Despite this prediction, mutations in the serotonin biosynthesis genes had little or modest effects on lifespan, while the mod-5 mutation with increased availability of serotonin caused a modest life-shortening effect. In contrast, a deletion mutation of the ser-1 serotonin receptor gene increased longevity by up to 46%, likely through the insulin/insulin-like growth factor 1 pathway. This result suggests an interaction between the serotonin pathway and the insulin/insulin-like growth factor 1 pathway. A deletion mutation of another serotonin receptor gene, ser-4 , shortened early to mid lifespan. The results suggest that serotonin signal antagonistically modulates longevity through different serotonin receptors. This study may indicate serotonin receptors as a potential target for antigeric interventions.     

Bidirectional regulation of thermotaxis by glutamate transmissions in Caenorhabditis elegans

In complex neural circuits of the brain, massive information is processed with neuronal communication through synaptic transmissions. It is thus fundamental to delineate information flows encoded by various kinds of transmissions. Here, we show that glutamate signals from two distinct sensory neurons bidirectionally affect the same postsynaptic interneuron, thereby producing the opposite behaviours. EAT-4/VGLUT (vesicular glutamate transporter)-dependent glutamate signals from AFD thermosensory neurons inhibit the postsynaptic AIY interneurons through activation of GLC-3/GluCl inhibitory glutamate receptor and behaviourally drive migration towards colder temperature. By contrast, EAT-4-dependent glutamate signals from AWC thermosensory neurons stimulate the AIY neurons to induce migration towards warmer temperature. Alteration of the strength of AFD and AWC signals led to significant changes of AIY activity, resulting in drastic modulation of behaviour. We thus provide an important insight on information processing, in which two glutamate transmissions encoding opposite information flows regulate neural activities to produce a large spectrum of behavioural outputs.     

CDK-5 regulates the abundance of GLR-1 glutamate receptors in the ventral cord of Caenorhabditis elegans

The proline-directed kinase Cdk5 plays a role in several aspects of neuronal development. Here, we show that CDK-5 activity regulates the abundance of the glutamate receptor GLR-1 in the ventral cord of Caenorhabditis elegans and that it produces corresponding changes in GLR-1-dependent behaviors. Loss of CDK-5 activity results in decreased abundance of GLR-1 in the ventral cord, accompanied by accumulation of GLR-1 in neuronal cell bodies. Genetic analysis of cdk-5 and the clathrin adaptin unc-11 AP180 suggests that CDK-5 functions prior to endocytosis at the synapse. The scaffolding protein LIN-10/Mint-1 also regulates GLR-1 abundance in the nerve cord. CDK-5 phosphorylates LIN-10/Mint-1 in vitro and bidirectionally regulates the abundance of LIN-10/Mint-1 in the ventral cord. We propose that CDK-5 promotes the anterograde trafficking of GLR-1 and that phosphorylation of LIN-10 may play a role in this process.     

Heterologous expression of functional G-protein-coupled receptors in Caenorhabditis elegans

New strategies for expression, purification, functional characterization, and structural determination of membrane-spanning G-protein-coupled receptors (GPCRs) are constantly being developed because of their importance to human health. Here, we report a Caenorhabditis elegans heterologous expression system able to produce milligram amounts of functional native and engineered GPCRs. Both bovine opsin [(b)opsin] and human adenosine A(2A) subtype receptor [(h)A(2A)R] expressed in neurons or muscles of C. elegans were localized to cell membranes. Worms expressing these GPCRs manifested changes in motor behavior in response to light and ligands, respectively. With a newly devised protocol, 0.6-1 mg of purified homogenous 9-cis-retinal-bound bovine isorhodopsin [(b)isoRho] and ligand-bound (h)A(2A)R were obtained from C. elegans from one 10-L fermentation at low cost. Purified recombinant (b)isoRho exhibited its signature absorbance spectrum and activated its cognate G-protein transducin in vitro at a rate similar to native rhodopsin (Rho) obtained from bovine retina. Generally high expression levels of 11 native and mutant GPCRs demonstrated the potential of this C. elegans system to produce milligram quantities of high-quality GPCRs and possibly other membrane proteins suitable for detailed characterization.     

Ionotropic glutamate receptors mediate juvenile hormone synthesis in the cockroach,Diploptera punctata

By monitoring changes in the cytosolic [Ca2+](i) and rates of juvenile hormone (JH) synthesis in response to L-glutamate agonists and antagonists, we identified and characterized glutamate receptor subtypes in corpus allatum (CA) cells of the cockroach, Diploptera punctata. During the first ovarian cycle, corpora allata exhibited a cycle of changes in sensitivity to L-glutamate correlated to cyclic changes in rates of JH synthesis. When exposed to 60 microM L-glutamate in vitro, the active corpora allata of day-4 mated females produced 60% more JH, while inactive corpora allata at other ages showed 10-20% stimulatory response. Pharmacological characterization using various L-glutamate receptor agonists and antagonists indicated that several ionotropic subtypes of L-glutamate receptors were present in the CA. The CA showed an increase in rates of JH synthesis in response to NMDA, kainate, and quisqualate, but not to AMPA in both L-15 medium and minimum incubation medium. In contrast, applications of the metabotropic receptor-specific agonist trans-ACPD failed to elicit a change in the cytosolic [Ca2+](i) and JH production.An elevation of cytosolic calcium concentration, followed by 20-30% rise in JH production, was observed when active CA cells were exposed to 10-40 microM kainate. Kainate had no stimulatory effect on JH synthesis in calcium-free medium. The kainate-induced JH synthesis was blocked by 20 microM CNQX but was not affected by 20 microM NBQX. Kainate-stimulated JH production was not suppressed by MK-801 (a specific blocker of NMDA-receptor channel), nor was NMDA-stimulated JH production affected by CNQX (a specific antagonist of kainate receptor). These data suggest that active CA cells are stimulated to synthesize more JH by a glutamate-induced calcium rise via NMDA-, kainate- and/or quisqualate-sensitive subtypes of ionotropic L-glutamate receptors. The metabotropic-subtype and ionotropic AMPA-subtype L-glutamate receptors are unlikely to be present on active CA cells.     

Functional expression of mammalian bitter taste receptors in Caenorhabditis elegans

Bitter taste has evolved as a central warning signal against the ingestion of potentially toxic substances appearing in the environment. The molecular events in the perception of bitter taste start with the binding of specific water-soluble molecules to G protein-coupled receptors (GPCR) called T2Rs and expressed at the surface of taste receptor cells. The functional characterisation of T2R receptors is far from been completed due to the difficulty to functionally express them in heterologous systems. Taking advantage of the parallelisms between the Caenorhabditis elegans (C. elegans) and mammalian GPCR signalling pathways, we developed a C. elegans-based expression system to express functional human and rodent GPCRs of the T2R family. We generated transgenic worms expressing T2Rs in ASI chemosensory neurons and performed behavioural assays using a variety of bitter tastants. As a proof of the concept, we generated transgenic worms expressing human T2R4 or its mouse ortholog T2R8 receptors, which respond to two bitter tastants previously characterised as their functional ligands, 6-n-propyl-2-thiouracil and denatoniun. As expected, expression of human T2R4 or its mouse ortholog T2R8 in ASI neurons counteracted the water-soluble avoidance to 6-n-propyl-2-thiouracil and denatoniun observed in control wild-type worms. The expression in ASI neurons of human T2R16, the ligand of which, phenyl-beta-d-glucopyranoside, belong to a chemically different group of bitter tastants, also counteracted the water-soluble avoidance to this compound observed in wild-type worms. These results indicate that C. elegans is a suitable heterologous expression system to express functional T2Rs providing a tool to efficiently search for specific taste receptor ligands and to extend our understanding of the molecular basis of gustation.     

The avermectin receptors of Haemonchus contortus and Caenorhabditis elegans

Most of the recent evidence suggests that the avermectin/milbemycin family of anthelmintics act via specific interactions with glutamate-gated chloride channels. These channels are encoded by a small family of genes in nematodes, though the composition of the gene family and the function of the individual members of the family may vary between species. We review our current knowledge concerning the properties of the glutamate-gated chloride channels from Caenorhabditis elegans and the related parasite, Haemonchus contortus. We conclude that the biological effects of the avermectins/milbemycins can be largely explained by the known pharmacology and distribution of the glutamate-gated chloride channels and that differences between the glutamate-gated chloride channels from different nematodes may underlie species-specific variations in anthelmintic action.     

Characterization of the Caenorhabditis elegans G protein-coupled serotonin receptors

Serotonin (5-HT) regulates a wide range of behaviors in Caenorhabditis elegans, including egg laying, male mating, locomotion and pharyngeal pumping. So far, four serotonin receptors have been described in the nematode C. elegans, three of which are G protein-coupled receptors (GPCR), (SER-1, SER-4 and SER-7), and one is an ion channel (MOD-1). By searching the C. elegans genome for additional 5-HT GPCR genes, we identified five further genes which encode putative 5-HT receptors, based on sequence similarities to 5-HT receptors from other species. Using loss-of-function mutants and RNAi, we performed a systematic study of the role of the eight GPCR genes in serotonin-modulated behaviors of C. elegans (F59C12.2, Y22D7AR.13, K02F2.6, C09B7.1, M03F4.3, F16D3.7, T02E9.3, C24A8.1). We also examined their expression patterns. Finally, we tested whether the most likely candidate receptors were able to modulate adenylate cyclase activity in transfected cells in a 5-HT-dependent manner. This paper is the first comprehensive study of G protein-coupled serotonin receptors of C. elegans. It provides a direct comparison of the expression patterns and functional roles for 5-HT receptors in C. elegans.Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Functional analysis of Caenorhabditis elegans glutamate receptor subunits by domain transplantation

Glutamate receptors are not only abundant and important mediators of fast excitatory synaptic transmission in vertebrates, but they also serve a similar function in invertebrates such as Drosophila and the nematode Caenorhabditis elegans. In C. elegans, an animal with only 302 neurons, 10 different glutamate receptor subunits have been identified and cloned. To study the ion channel properties of these receptor subunits, we recorded glutamate-gated currents from Xenopus oocytes that expressed either C. elegans glutamate receptor subunits or chimeric rat/C. elegans glutamate receptor subunits. The chimeras were constructed between the C. elegans glutamate receptor pore domains and either the rat kainate receptor subunit GluR6, the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor subunit GluR1, or the N-methyl-d-aspartate (NMDA) receptor subunit NMDAR1-1a. Although native subunits were nonfunctional, 9 of 10 ion pores were found to conduct current upon transplantation into rat receptor subunits. A provisional classification of the C. elegans glutamate receptor subunits was attempted based on functionality of the chimeras. C. elegans glutamate receptor ion pores, at a position homologous to a highly conserved site critical for ion permeation properties in vertebrate glutamate receptor pores, contain amino acids not found in vertebrate glutamate receptors. We show that the pore-constricting Q/R site, which in vertebrate receptors determines calcium permeability and rectification properties of the ion channel, in C. elegans can be occupied by other amino acids, including, surprisingly, lysine and proline, without loss of these properties.     

Ionotropic Receptors (IRs): Chemosensory ionotropic glutamate receptors in Drosophila and beyond

Ionotropic Receptors (IRs) are a recently characterized family of olfactory receptors in the fruit fly, Drosophila melanogaster. IRs are not related to insect Odorant Receptors (ORs), but rather have evolved from ionotropic glutamate receptors (iGluRs), a conserved family of synaptic ligand-gated ion channels. Here, we review the expression and function of IRs in Drosophila, highlighting similarities and differences with iGluRs. We also briefly describe the organization of the neuronal circuits in which IRs function, comparing and contrasting them with the sensory pathways expressing ORs. Finally, we summarize the bioinformatic identification and initial characterization of IRs in other species, which imply an evolutionarily conserved role for these receptors in chemosensation in insects and other protostomes.     

Ionotropic glutamate receptors in the external lateral parabrachial nucleus participate in processing cardiac sympathoexcitatory reflexes

Stimulation of cardiac sympathetic afferents during myocardial ischemia with metabolites such as bradykinin (BK) evokes sympathoexcitatory reflex responses and activates neurons in the external lateral parabrachial nucleus (elPBN). The present study tested the hypothesis that this region in the pons processes sympathoexcitatory cardiac reflexes through an ionotropic glutamate receptor mechanism. The ischemic metabolite BK (0.1-1 μg) was injected into the pericardial space of anesthetized and bilaterally vagotomized or intact cats. Hemodynamic and renal sympathetic nerve activity (RSNA) responses to repeated administration of BK before and after unilateral 50-nl microinjections of kynurenic acid (Kyn; 25 mM), 2-amino-5-phosphonopentanoic acid (AP5; 25 mM), and 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzol(F)quinoxaline (NBQX; 10 mM) into the elPBN were recorded. Intrapericardial BK evoked significant increases in mean arterial pressure (MAP) and RSNA in seven vagotomized cats. After blockade of glutamate receptors with the nonselective glutamate receptor antagonist Kyn, the BK-evoked reflex increases in MAP (50 ± 6 vs. 29 ± 2 mmHg) and RSNA (59 ± 8.6 vs. 29 ± 4.7%, before vs. after) were significantly attenuated. The BK-evoked responses returned to pre-Kyn levels 85 min after the application of Kyn. Similarly, BK-evoked reflex responses were reversibly attenuated by blockade of glutamate N-methyl-d-aspartate (NMDA) receptors with AP5 (n = 5) and α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors with NBQX (n = 5). In contrast, we observed that the repetitive administration of BK evoked consistent reflex responses including MAP and RSNA before and after microinjection of 50 nl of the artificial cerebrospinal fluid vehicle into the elPBN in five animals. Microinjection of glutamate receptor antagonists into regions outside the elPBN did not alter BK-induced reflex responses. Microinjection of Kyn into the elPBN reversibly attenuated BK-induced reflex responses in four vagus intact animals. These data are the first to show that NMDA and AMPA ionotropic glutamate receptors in the elPBN play an important role in processing cardiac excitatory reflex responses.     

A membrane associated glutamate binding protein from Caenorhabditis elegans and Haemonchus contortus

1. A glutamate binding protein has been identified in membrane preparations from the free living nematode, Caenorhabditis elegans, and from the parasitic nematode, Haemonchus contortus. 2. This putative glutamate receptor was solubilized with 30 mM octyl-B-glucoside and partially purified by anion exchange and gel filtration chromatography. 3. An 80-fold purification with recovery of 75% of the glutamate binding activity was achieved. 4. The soluble C. elegans binding protein displayed a Kd for glutamate of 0.1 microM, in close agreement with the findings for the membrane associated binding protein. 5. Quisqualate was capable of displacing glutamate from the soluble C. elegans receptor, again in agreement with previous findings for the membrane bound receptor. 6. The fact that a parasitic nematode, Haemonchus contortus, also possesses this putative glutamate receptor, strengthens the case for using C. elegans as a model system for the study of parasitic nematode neuromuscular physiology.