Glutamate Receptor Channels Research Articles

Primary structure and expression of the γ2 subunit of the glutamate receptor channel selective for kainate

The presence and primary structure of a novel subunit of the mouse glutamate receptor channel, designated as γ2, have been revealed by cloning and sequencing the cDNA. The γ2 subunit has structural characteristics common to the neurotransmitter-gated ion channel family and shares a high amino acid sequence identity with the rat KA-1 subunit, thus constituting the γ subfamily of the glutamate receptor channel. Expression of the γ2 subunit together with the β2 subunit in Xenopus oocytes yields functional glutamate receptor channels selective for kainate.

Structural determinants of barium permeation and rectification in non- NMDA glutamate receptor channels

Structural determinants of barium permeation and rectification in non- NMDA glutamate receptor channels

Functional experession of the α1 subunit of the AMPA-selective glutamate receptor channel, using a baculovirus system

Using a baculovirus expression vector system, the α1 subunit of the mouse AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionate)-selective glutamate receptor channel was expressed in insect Spodoptera frugiperda cells. Binding studies using [ 3H]AMPA showed that insect cells infected with the recombinant virus expressed approximately 1.8×10 5 binding sites per cell on their surface. The ligand binding characteristics of the receptors expressed in insect cells were examined. The baculovirus-insect cell expression system affords high-efficiency expression of the receptor in sufficient amounts to permit structural and functional analyses.

RNA editing in brain controls a determinant of ion flow in glutamate-gated channels

l-glutamate, the principal excitatory transmitter in the brain, gates ion channels mediating fast neurotransmission. Subunit components of two related classes of glutamate receptor channels have been characterized by cDNA cloning and shown to carry either an arginine or a glutamine residue in a defined position of their putative channel-forming segment. The arginine residue in this segment profoundly alters, and dominates, the properties of ion flow, as demonstrated for one channel class. We now show that the genomic DNA sequences encoding the particular channel segment of all subunits harbor a glutamine codon (CAG), even though an arginine codon (CGG) is found in mRNAs of three subunits. Multiple genes and alternative exons were excluded as sources for the arginine codon; hence, we propose that transcripts for three subunits are altered by RNA editing. This process apparently edits subunit transcripts of the two glutamate receptor classes with different efficiency and selectivity.

A single amino acid residue determines the Ca2+ permeability of AMPA-selective glutamate receptor channels

Functional analysis of AMPA-selective glutamate receptor channels expressed in Xenopus oocytes from cloned cDNAs has shown that homomeric channels formed by the GluR1 subunit are permeable to Ca2+, whereas heteromeric channels composed of the GluR1 and GluR2 subunits show little permeability. Furthermore, substitution of glutamine for arginine in putative transmembrane segment M2 of the GluR2 subunit makes the heteromeric channels permeable to Ca2+. These results suggest that the GluR2 subunit plays a key role in keeping AMPA-selective glutamate receptor channels essentially impermeable to Ca2+ and that the critical determinant is the positively charged residue in M2 segment.

Enhancement of the Glutamate Response by cAMP-Dependent Protein Kinase in Hippocampal Neurons

Receptor channels activated by glutamate, an excitatory neurotransmitter in the mammalian brain, are involved in processes such as long-term potentiation and excitotoxicity. Studies of glutamate receptor channels expressed in cultured hippocampal pyramidal neurons reveal that these channels are subject to neuromodulatory regulation through the adenylate cyclase cascade. The whole-cell current response to glutamate and kainate [a non-NMDA (N-methyl-D-aspartate) receptor agonist] was enhanced by forskolin, an activator of adenylate cyclase. Single-channel analysis revealed that an adenosine 3',5'-monophosphate-dependent protein kinase (PKA) increases the opening frequency and the mean open time of the non-NMDA-type glutamate receptor channels. Analysis of synaptic events indicated that forskolin, acting through PKA, increased the amplitude and decay time of spontaneous excitatory postsynaptic currents.

Excitatory amino acid receptor-channels in Purkinje cells in thin cerebellar slices

Glutamate receptors of the N-methyl-D-aspartate (NMDA) and non-NMDA type serve different functions during excitatory synaptic transmission. Although many central neurons bear both types of receptor, the evidence concerning the sensitivity of cerebellar Purkinje cells to NMDA is contradictory. To investigate the receptor types present in Purkinje cells, we have used whole-cell and outside-out patch-clamp methods to record from cells in thin cerebellar slices from young rats. At a holding potential of -70 mV (in nominally Mg(2+)-free medium, with added glycine) NMDA caused a whole-cell current response which consisted of a dramatic increase in the frequency of synaptic currents. In the presence of tetrodotoxin (TTX) and the gamma-aminobutyric acidA (GABAA) receptor antagonist bicuculline, spontaneous synaptic currents and responses to NMDA were inhibited. In a proportion of cells a small polysynaptic response to NMDA persisted, which was further reduced by the non-NMDA receptor antagonist 6-cyano-2,3-dihydro-7-nitroquinoxalinedione (CNQX). The non-NMDA glutamate receptor agonists kainate (KA), quisqualate (QA) and s-alpha-amino-3-hydroxy-5-methyl-4-isoazolepropionic acid (s-AMPA), evoked large inward currents due to the direct activation of receptors in Purkinje cells. NMDA applied to excised membrane patches failed to evoke any single-channel currents, whereas s-AMPA and QA caused small inward currents accompanied by marked increases in current noise. Spectral analysis of the s-AMPA noise in patches gave an estimated mean channel conductance of approximately 4 pS.(ABSTRACT TRUNCATED AT 250 WORDS)

Receptors coupled to ionic channels: the glutamate receptor family

Glutamate-gated ion channels belong to a complex family of receptors containing several pharmacological subtypes. They are thought to be essential for the acquisition of associative memory and for activity-dependent synaptogenesis, and have been implicated in several central nervous system diseases. Within the past year, molecular cloning of the first glutamate receptor channel and several related subunits has opened new approaches for understanding the basis of these important phenomena.

特集 イオンチャネルとリセプター 中枢ニューロンのグルタミン酸受容体チャネル

The amino acid glutamate is a major excitatory neurotransmitter in the mammalian central nervous system. It has been proposed that the excitatory actions of this neurotransmitter are mediated mainly by three pharmacologically distinct classes of receptor channels. They are named according to their selective agonists as the NMDA (N-methyl-D-aspartate), AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionate), and kainate receptors. The NMDA receptor is characterized by voltage-dependent block by Mg2+, high Ca2+ permeability, and potentiation by glycine, being obviously distinct from AM PA and kainate receptors. On the other hand, it has been difficult to distinguish AMPA and kainate receptors. Molecular genetic studies have recently provided evidence that AMPA and kainate activate the same receptor molecules, and that a variety of structurally and functionally different receptor channels can be assembled from multiple KA/AMPA receptor subunits. In line with these studies, we have revealed the presence of two distinct types of KA/AMPA responses which differ in their rectification properties and their permeabilities to Ca2+ in cultured rat hippocampal neurons. In this article, I review recent progress in research on glutamate receptor channels in the mammalian central nervous system.

Currents through single glutamate receptor channels in outside‐out patches from rat cerebellar granule cells.

1. Single-channel currents evoked in outside-out membrane patches from rat cerebellar granule cells by glutamate, aspartate, N-methyl-D-aspartate (NMDA), kainate and quisqualate were studied. Each agonist produced openings to five discrete amplitude levels. At a membrane potential of -70 mV, these levels correspond to single-channel conductances of about 8, 17, 30, 40 and 50 pS. NMDA, aspartate and glutamate evoked mainly 50 pS openings and also substantial numbers of 40 pS events. Kainate evoked primarily 8 and 17 pS openings. 2. The relative proportion of openings to each conductance level showed no dependence on membrane potential. At membrane potentials negative to -100 mV, current-voltage plots for 30, 40 and 50 pS openings showed substantial inward rectification. 3. With NMDA, aspartate and glutamate, the most common type of direct transition was between the 50 pS open level and the shut level. Transitions between the 30, 40 and 50 pS levels were also relatively common. With few exceptions, 8 and 17 pS openings appeared to arise directly from, and return directly to, the shut level. The differences between granule cells and certain other central neurones, in the types of transitions associated with NMDA receptor channels, provide evidence for the existence of more than one type of NMDA receptor. 4. Four exponential components were identified consistently in the shut-time distributions that were obtained with NMDA, aspartate and glutamate. Mean time constants for the briefest two components were 30 to 65 microseconds and 0.65 to 1.00 ms. The mean duration of these 'gaps within bursts' differed for different agonists, but did not vary with membrane potential. 5. Two exponential components were distinguished in most open-time distributions for the 50 pS level (time constants, 0.9-1.2 and 3.2-3.9 ms at -100 mV), whereas open-time distributions for 30 and 40 pS events were described adequately by single exponentials with time constants below 1.0 ms. The duration of 50 pS openings decreased with hyperpolarization. 6. Mean open times for 8 and 17 pS events produced by NMDA, aspartate and glutamate were 0.3 to 0.7 ms. The longest such openings were observed with quisqualate. 7. Three exponential components were present in distributions of burst length, and of total open time per burst, that were obtained with NMDA, aspartate and glutamate. The slowest two burst-length components had mean time constants of 1.7-2.4 and 10.6-13.0 ms and originated from the kinetic behaviour of the 50 pS state.(ABSTRACT TRUNCATED AT 400 WORDS)

Molecular biology of the glutamate receptor channel

Molecular biology of the glutamate receptor channel

Potentiation of glutamate-activated currents in isolated hippocampal neurons

“Fast chemical stimulation” was shown to induce potentiation of glutamate-activated currents in neurons isolated from rat hippocampus. A fast application system allowed solution changes up to a rate of 20 Hz. In Mg 2+-free solution, the response to glutamate application immediately after repetitive stimulation with glutamate plus glycine was increased by 25%–88%, returning to control levels over 10–15 min. Enhancement of glutamate-induced currents was also seen after stimulation with solutions containing aspartate or NMDA plus glycine. Aspartate-induced currents were not potentiated. These and other observations demonstrate that in a purely “postsynaptic” system, short-term potentiation can be induced and is mediated via NMDA receptors whereas the potentiated current is carried via non-NMDA glutamate receptor channels.

Glutamate receptor channels in rat DRG neurons: Activation by kainate and quisqualate and blockade of desensitization by con A

Primary afferent C fibers in rat dorsal roots are depolarized by the excitatory amino acids kainate and domoate. Under whole-cell voltage clamp, kainate and domoate increase membrane conductance in a subpopulation of freshly dissociated DRG neurons. In contrast to kainate currents observed in CNS neurons, responses to kainate and domoate in DRG cells desensitize with prolonged agonist exposure. Half-maximal activation is achieved with much lower concentrations of kainate and domoate in sensory neurons than in CNS neurons from cerebral cortex. Rapid applications of glutamate, quisqualate, and AMPA evoke a transient current in DRG neurons and desensitize cells to subsequent applications of kainate or domoate. Brief incubation with the lectin concanavalin A eliminates desensitization to excitatory amino acids; after treatment with concanavalin A, all five agonists gate sustained currents of similar amplitude via the same receptor.

Glutamate receptor-channel gating. Maximum likelihood analysis of gigaohm seal recordings from locust muscle

Gigaohm recordings have been made from glutamate receptor channels in excised, outside-out patches of collagenase-treated locust muscle membrane. The channels in the excised patches exhibit the kinetic state switching first seen in megaohm recordings from intact muscle fibers. Analysis of channel dwell time distributions reveals that the gating mechanism contains at least four open states and at least four closed states. Dwell time autocorrelation function analysis shows that there are at least three gateways linking the open states of the channel with the closed states. A maximum likelihood procedure has been used to fit six different gating models to the single channel data. Of these models, a cooperative model yields the best fit, and accurately predicts most features of the observed channel gating kinetics.

Markov, fractal, diffusion, and related models of ion channel gating. A comparison with experimental data from two ion channels

The gating kinetics of single-ion channels are generally modeled in terms of Markov processes with relatively small numbers of channel states. More recently, fractal (Liebovitch et al. 1987. Math. Biosci. 84:37-68) and diffusion (Millhauser et al. 1988. Proc. Natl. Acad. Sci. USA. 85:1502-1507) models of channel gating have been proposed. These models propose the existence of many similar conformational substrates of the channel protein, all of which contribute to the observed gating kinetics. It is important to determine whether or not Markov models provide the most accurate description of channel kinetics if progress is to be made in understanding the molecular events of channel gating. In this study six alternative classes of gating model are tested against experimental single-channel data. The single-channel data employed are from (a) delayed rectifier K+ channels of NG 108-15 cells and (b) locust muscle glutamate receptor channels. The models tested are (a) Markov, (b) fractal, (c) one-dimensional diffusion, (d) three-dimensional diffusion, (e) stretched exponential, and (f) expo-exponential. The models are compared by fitting the predicted distributions of channel open and closed times to those observed experimentally. The models are ranked in order of goodness-of-fit using a boot-strap resampling procedure. The results suggest that Markov models provide a markedly better description of the observed open and closed time distributions for both types of channel. This provides justification for the continued use of Markov models to explore channel gating mechanisms.

Whole‐cell current noise produced by excitatory and inhibitory amino acids in large cerebellar neurones of the rat.

1. Membrane noise and current changes produced by glutamate and related excitatory amino acids have been examined in cultured large cerebellar neurones (including Purkinje cells), with whole-cell patch-clamp methods. The sensitivity of these neurones to the inhibitory amino acids gamma-aminobutyric acid (GABA) and glycine has also been studied. 2. The neurones formed inhibitory synapses in culture, and displayed spontaneous synaptic currents. Reducing the pipette Cl- concentration (i.e. intracellular synaptic currents. Reducing the pipette Cl- concentration (i.e. intracellular concentration) caused a negative shift in their reversal potential, and the currents could be blocked with bicuculline (10 microM), suggesting that they were mediated by GABAA receptors. Spontaneous synaptic activity was also considerably reduced in the presence of 3 microM-tetrodotoxin. 3. Analysis of the increase in whole-cell current noise produced by the application of GABA (3 microM) gave noise spectra that were fitted by two Lorentzian components with slow and fast time constants of 23.6 and 1.9 ms at a membrane potential (Vm) of -110 mV. The mean single-channel conductance estimated from GABA noise was gamma noise = 12 pS. Glycine (10 microM) whole-cell current responses were Cl(-)-mediated and reversibly abolished by 1 microM-strychnine. 4. Bath application of excitatory amino acids gave whole-cell current changes accompanied by an increase in synaptic activity. Postsynaptic responses to the excitatory amino acids were more readily seen after the inhibitory synaptic currents had been abolished by bicuculline. Membrane current changes were obtained in response to the putative transmitters glutamate and aspartate, and the agonists NMDA (N-methyl-D-aspartate), ibotenate, quisqualate and kainate. Their reversal potential was approximately -5 mV. 5. A majority of noise spectra produced by the various glutamate receptor agonists were fitted by two Lorentzian components; the rest were fitted with a single Lorentzian component. The noise time constants were apparently not dependent on the type of glutamate agonist used to activate the receptor channels. Pooling data for all agonists gave a mean time constant for single-component spectra of tau noise = 4.8 +/- 0.3 ms; for two-component spectra the time constants were tau 1 = 22.7 +/- 1.8 ms and tau 2 = 2.2 +/- 0.12 ms (Vm = -110 to -50 mV). It is likely that the two components present in whole-cell noise spectra reflect complex kinetics of glutamate receptor channels. 6. The mean single-channel conductance was estimated from whole-cell noise for the various excitatory amino acids.(ABSTRACT TRUNCATED AT 400 WORDS)

Synthesis and receptor binding studies of (+/-)1-iodo-MK-801.

The glutamate analogue N-methyl-D-aspartate (NMDA) binds to a subset of glutamate receptors that are coupled to a voltage-sensitive cation channel. This NMDA-linked channel is the likely binding locus of the potent anticonvulsant MK-801. To develop single-photon emission computed tomography (SPECT) probes of this brain channel, we synthesized (+/)1-iodo-MK-801 and (+/-)1-[125I]iodo-MK-801. The effect of (+/-)1-iodo-MK-801 on ligand binding to the NMDA-linked glutamate receptor site was assessed using a rat brain homogenate assay. (+/-)1-Iodo-MK-801 displaced the dissociative anesthetic ligand [3H]N-[1-(2-thienyl)cyclohexyl]piperidine ([3H]TCP) binding with an IC50 of 1 microM, which is a 10-fold lower binding affinity than that of (+/-)MK-801. In in vivo autoradiographic studies, (+/-)MK-801 failed to block selective uptake of (+/-)1-iodo-MK-801 in rat brain. These results suggest that (+/-)1-iodo-MK-801 may not be a suitable ligand for mapping NMDA-linked glutamate receptor channels.

Ca-channels and glutamate receptor-channel of cultured chick cochlear ganglionic neuron

Ca-channels and glutamate receptor-channel of cultured chick cochlear ganglionic neuron

The use of dwell time cross-correlation functions to study single-ion channel gating kinetics

The derivation of cross-correlation functions from single-channel dwell (open and closed) times is described. Simulation of single-channel data for simple gating models, alongside theoretical treatment, is used to demonstrate the relationship of cross-correlation functions to underlying gating mechanisms. It is shown that time irreversibility of gating kinetics may be revealed in cross-correlation functions. Application of cross-correlation function analysis to data derived from the locust muscle glutamate receptor-channel provides evidence for multiple gateway states and time reversibility of gating. A model for the gating of this channel is used to show the effect of omission of brief channel events on cross-correlation functions.

ChemInform Abstract: The Total Synthesis of Argiopine (Argiotoxin‐636).

Abstract The total synthesis of a constituent of spider venom with reported glutamate receptor channel activity is described.