Mechanisms of the blockade of glutamate channel receptors: Significance for structural and physiological investigations

The mechanism of the blocking action of phenylcyclohexyl derivative IEM-1925 on ionotropic NMDA and AMPA glutamate receptors was studied. Experiments on isolated rat brain neurons (hippocampal pyramidal cells and striatal cholinergic interneurons) were performed using local voltage clamping in the “whole cell” configuration. In equilibrium conditions at a membrane potential of −80 mV, there was no selectivity in the action of IEM-1925 on the open channels of either type of glutamate receptor. However, data were obtained showing significant differences in the mechanisms of the blocking actions. Although IEM-1925 was unable to penetrate into closed channels of either receptor type, molecules were able to leave closed AMPA receptor channels but not closed NMDA receptor channels. In hyperpolarization, the departure of the blocker from open NMDA receptor channels was slowed, while departure from open and closed AMPA receptor channels was accelerated. The blocker thus appeared able to penetrate AMPA receptor channels to enter cells, the gating mechanism of these channels being located above the blocker binding site. The actions of IEM-1925 on NMDA and AMPA receptors were compared with its ability to suppress tremor in mice induced with s.c. doses of arecoline. The results indicated that both types of receptors have a role in producing tremor. The differences in the mechanisms of action on AMPA and NMDA receptors may explain the ambiguous nature of the effects of the glutamate channel blocker in experimental therapy. __________ Translated from Rossiiskii Fiziologicheskii Zhurnal imeni I. M. Sechenova, Vol. 92, No. 1, pp. 27–38, January, 2006.     

Maximum likelihood fitting of single channel NMDA activity with a mechanism composed of independent dimers of subunits

Steady-state single channel activity from NMDA receptors was recorded at a range of concentrations of both glutamate and glycine. The results were fitted with several plausible mechanisms that describe both binding and gating. The mechanisms we have tested were based on our present understanding of receptor structure, or based on previously proposed mechanisms for these receptors. The steady-state channel properties appear to have virtually no dependence on the concentration of either ligand, other than the frequency of channel activations. This limited the ability to discriminate detail in the mechanism, and, along with the persistence of open–shut correlations in high agonist concentrations, suggests that NMDA channels, unlike other neurotransmitter receptors, cannot open unless all binding sites are occupied. As usual for analyses of NMDA channels, the applicability of our results to physiological observations is limited by uncertainties in synaptic zinc and hydrogen ion concentrations, both of these being known to affect the receptor. The mechanism that we propose, on the basis of steady-state single channel recordings, predicts with fair accuracy the apparent open and shut-time distributions in different concentrations of agonists, correlations between open and shut times, and both the rising and falling phases of the macroscopic response to concentration jumps, and can therefore account for the main features of synaptic currents.     

Intracellular spermine decreases open probability of N-methyl-D-aspartate receptor channels

Spermine and related polyamines have been shown to be endogenous regulators of several ion channel types including ionotropic glutamate receptors. The effect of spermine on N-methyl-d-aspartate (NMDA) receptors in cultured rat hippocampal neurons was studied using single-channel and whole-cell patch clamp recordings. Intracellular spermine resulted in the dose-dependent inhibition of NMDA-induced responses. Spermine reversibly inhibited the single NMDA receptor channel activity in inside-out patches suggesting a membrane-delimited mechanism of action. Open probability of NMDA receptor channels was decreased in a dose-dependent manner. Mechanism of spermine-induced inhibition of NMDA receptor was different from that of intracellular Ca(2+)-induced NMDA receptor inactivation. Both pharmacological studies and single channel analysis indicate that in contrast to the effect of extracellular spermine the intracellular spermine effect is not dependent on the NMDA receptor subunit composition. We propose that intracellular spermine has a direct inhibitory effect on NMDA receptors that is different from calcium-induced NMDA receptor inactivation and spermine-induced voltage-dependent inhibition of AMPA/kainate receptors. Spermine-induced tonic change in the open probability of NMDA receptor channels may play a role in mechanisms underlying short-term changes in the synaptic efficacy.     

Distinct gating modes determine the biphasic relaxation of NMDA receptor currents

Following brief stimulation, macroscopic NMDA receptor currents decay with biphasic kinetics that is believed to reflect glutamate dissociation and receptor desensitization. We found that the fast and slow decay components arise from the simultaneous deactivation of receptor populations that gate with short and long openings, respectively. Because individual receptors switched infrequently between gating modes, the relaxation time course was largely determined by the proportion of channels in each gating mode at the time of stimulation.     

Inhibition of glutamate transporters couples to Kv4.2 dephosphorylation through activation of extrasynaptic NMDA receptors

Activation of glutamate receptors is known to modulate K⁺ channel surface trafficking, phosphorylation, and function, and increasing evidence has implicated K⁺ channels in plastic changes in glutamatergic synapses. Kv4.2 channels control the amplitude of back-propagating action potentials and shape postsynaptic responses in hippocampus, and synaptic glutamate receptor activation leads to increased phosphorylation of Kv4.2 channels that is associated with enhanced synaptic plasticity. Thus, we investigated the possibility that activation of extrasynaptic NMDA-type glutamate receptors couples to Kv4.2 channel dephosphorylation. In hippocampal neurons, we found that selective activation of extrasynaptic NMDA receptors dephosphorylates Kv4.2 channels, and driving synaptic activity increases phosphorylation of Kv4.2. We also observed that Ca²⁺ entry through NMDA receptors is necessary for dephosphorylation of Kv4.2 channels. Consistent with a synaptic and extrasynaptic localization at hippocampal synapses, a fraction of Kv4.2 channel clusters was found to localize outside of pre- and postsynaptic markers. Excitatory amino acid transporters (EAATs) regulate ambient extracellular glutamate levels that active extrasynaptic NMDA receptors, and inhibition of glutamate uptake by blocking EAATs with the non-selective transporter inhibitor dl-threo-β-benzyloxyaspartic acid (TBOA) or the EAAT1/3 selective inhibitor l-serine O-sulfate (SOS) dephosphorylates Kv4.2 channels. These findings in conjunction with previous reports support the interesting possibility that synaptic and extrasynaptic NMDA receptors bi-directionally regulate phosphorylation levels of Kv4.2 channels in hippocampus. Moreover, we observed that EAAT activity controls extrasynaptic NMDA receptor modulation of Kv4.2 channel dephosphorylation.     

Glutamate receptor gating

Ionotropic glutamate receptors (iGluRs) mediate the vast majority of fast excitatory synaptic transmissions within the mammalian central nervous system (CNS). As for other ion channel protein families, there has been astounding progress in recent years in elucidating the details of protein structure through the crystallization of at least part of the ion channel protein complex. The result is a new framework for the interpretation of both classic and emerging functional data. Here we summarize, compare, and contrast recent findings for the AMPA, kainate, and NMDA subtypes of glutamate receptor ion channels, with an emphasis on the functional and structural aspects of how agonist binding controls channel gating.     

Protein kinase C modulates NMDA receptor trafficking and gating

Regulation of neuronal N-methyl-D-aspartate receptors (NMDARs) by protein kinases is critical in synaptic transmission. However, the molecular mechanisms underlying protein kinase C (PKC) potentiation of NMDARs are uncertain. Here we demonstrate that PKC increases NMDA channel opening rate and delivers new NMDA channels to the plasma membrane through regulated exocytosis. PKC induced a rapid delivery of functional NMDARs to the cell surface and increased surface NR1 immunofluorescence in Xenopus oocytes expressing NMDARs. PKC potentiation was inhibited by botulinum neurotoxin A and a dominant negative mutant of soluble NSF-associated protein (SNAP-25), suggesting that receptor trafficking occurs via SNARE-dependent exocytosis. In neurons, PKC induced a rapid delivery of functional NMDARs, assessed by electrophysiology, and an increase in NMDAR clusters on the surface of dendrites and dendritic spines, as indicated by immunofluorescence. Thus, PKC regulates NMDAR channel gating and trafficking in recombinant systems and in neurons, mechanisms that may be relevant to synaptic plasticity.     

Mutation of a glutamate receptor motif reveals its role in gating and delta2 receptor channel properties

Despite its importance in the cerebellum, the functions of the orphan glutamate receptor delta2 are unknown. We examined a mutant delta2 receptor channel in lurcher mice that was constitutively active in the absence of ligand. Because this mutation was within a highly conserved motif (YTANLAAF), we tested its effect on several glutamate receptors. Mutant delta2 receptors showed distinct channel properties, including double rectification of the current-voltage relationship, sensitivity to a polyamine antagonist and moderate Ca 2+ permeability, whereas other constitutively active mutant glutamate channels resembled wild-type channels in these respects. Moreover, the kinetics of ligand-activated currents were strikingly altered. We conclude that the delta2 receptor has a functional ion channel pore similar to that of glutamate receptors. The motif may have a role in the channel gating of glutamate receptors.     

Blocker Studies of the Functional Architecture of the NMDA Receptor Channel

Blockade of ion channels passing through the NMDA receptors of isolated rat hippocampus pyramidal neurons with tetraalkylammonium compounds, 9-aminoacridine, and Mg²⁺was studied using patch-clamp methods in the whole-cell configuration. Currents through NMDA channels were evoked by application of 100 M aspartate in magnesium-free medium containing glycine (3 M) to neurons. Analysis of the kinetics, charge transfer, and relationships between the extent of suppression of stationary currents on the one hand and membrane potential, agonist concentration, and blocker concentration on the other showed that blockers had different effects on the closing, desensitization, and agonist dissociation of NMDA channels. The size of the blocker was found to be the decisive factor determining its action on the gating functions of NMDA channels: larger blockers prevented closure and/or desensitization of the channel; smaller blockers only had partial effects on these processes, while the smallest blockers had no effect at all. These experiments showed that the apparent affinity of the blocker for the channel (1/IC₅₀) depended not only on the microscopic equilibrium dissociation constant (K _d), but also on the number of blocker binding sites, their mutual influences, and, of particular importance, the interaction of the blocker with the gating structures of the channel. These data led us to propose hypotheses relating to the geometry of the NMDA channel and the structure of its gating mechanism. The channel diameter at the level of activated gates was estimated to be 11 .     

NMDA receptor kinetics and synaptic function

Presynaptic release of L-glutamate mediates neurotransmission at most excitatory synapses in the vertebrate central nervous system. At the postsynaptic membrane, glutamate binds to two classes of ligand-gated ion channels, AMPA receptors and NMDA receptors. These channels are the basis of the two kinetically distinct components of the excitatory postsynaptic current (epsc). The slower synaptic conductance is mediated by NMDA receptor channels which, after binding glutamate, activate slowly and can remain activated for several hundred milliseconds. The average latency between glutamate binding and channel opening is at least several milliseconds and may be much longer. If the time to first opening is short many fewer channels would be required at each synaptic site to account for the amplitude of the NMDA receptor component of spontaneous miniature epscs, than if the time to first opening is very long.     

Phenylethanolamines inhibit NMDA receptors by enhancing proton inhibition

The phenylethanolamines, ifenprodil and CP-101,606, are NMDA receptor antagonists with promising neuroprotective properties. In recombinant NMDA receptors expressed in Xenopus oocytes, we found that these drugs inhibit NMDA receptors through a unique mechanism, making the receptor more sensitive to inhibition by protons, an endogenous negative modulator. These findings support a critical role for the proton sensor in gating the NMDA receptor and point the way to identifying a context-dependent NMDA receptor antagonist that is inactive at physiological pH, but is a potent inhibitor during the acidic conditions that arise during epilepsy, ischemia and brain trauma.     

Cytoplasmic and cytoskeletal regulation of glutamate receptor channels

The regulation of glutamate receptor channels by intracellular messengers is an important component in the plasticity of central excitatory synapses. This review summarizes recent evidence demonstrating that phosphorylation, intracellular calcium and interactions with cytoskeletal proteins can dramatically influence the properties of N-methyl-D-aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor channels. Kinases and intracellular calcium exert their action either directly on glutamate receptor channel subunits or indirectly via regulatory proteins or second messenger cascades. Recent evidence also suggests that interactions with cytoskeletal proteins play a dynamic role in regulating the gating of glutamate receptor channels. These studies provide tantalizing evidence for a complex set of regulatory interactions in the postsynaptic density.     

Functional NR2B- and NR2D-containing NMDA receptor channels in rat substantia nigra dopaminergic neurones

NMDA receptors regulate burst firing of dopaminergic neurones in the substantia nigra pars compacta (SNc) and may contribute to excitotoxic cell death in Parkinson's disease (PD). In order to investigate the subunit composition of functional NMDA receptors in identified rat SNc dopaminergic neurones, we have analysed the properties of individual NMDA receptor channels in outside-out patches. NMDA (100 n m ) activated channels corresponding to four chord conductances of 18, 30, 41 and 54 pS. Direct transitions were observed between all conductance levels. Between 18 pS and 41 pS conductance levels, direct transitions were asymmetric, consistent with the presence of NR2D-containing NMDA receptors. Channel activity in response to 100 n m or 200 μ m NMDA was not affected by zinc or TPEN ( N,N,N',N '-tetrakis-[2-pyridylmethyl]-ethylenediamine), indicating that SNc dopaminergic neurones do not contain functional NR2A subunits. The effect of the NR2B antagonist ifenprodil was complex: 1 μ m ifenprodil reduced open probability, while 10 μ m reduced channel open time but had no effect on open probability of channels activated by 100 n m NMDA. When the concentration of NMDA was increased to 200 μ m , ifenprodil (10 μ m ) produced the expected reduction in open probability. These results indicate that NR2B subunits are present in SNc dopaminergic neurones. Taken together, these findings indicate that NR2D and NR2B subunits form functional NMDA receptor channels in SNc dopaminergic neurones, and suggest that they may form a triheteromeric NMDA receptor composed of NR1/NR2B/NR2D subunits.     

Casein kinase-II regulates NMDA channel function in hippocampal neurons

Several second-messenger-regulated protein kinases have been implicated in the regulation of N-methyl-D-aspartate (NMDA) channel function. Yet the role of calcium and cyclic-nucleotide-independent kinases, such as casein kinase II (CKII), has remained unexplored. Here we identify CKII as an endogenous Ser/Thr protein kinase that potently regulates NMDA channel function and mediates intracellular actions of spermine on the channel. The activity of NMDA channels in cell-attached and inside-out recordings was enhanced by CKII or spermine and was decreased by selective inhibition of CKII. In hippocampal slices, inhibitors of CKII reduced synaptic transmission mediated by NMDA but not AMPA receptors. The dependence of NMDA receptor channel activity on tonically active CKII thus permits changes in intracellular spermine levels or phosphatase activities to effectively control channel function.     

Properties of vertebrate glutamate receptors: calcium mobilization and desensitization.

Glutamate is now recognized as a major excitatory neurotransmitter in the vertebrate CNS, participating in a number of physiological and pathological processes. The importance of glutamate in the mobilization of intracellular Ca2+ as well as the relationship between excitatory and toxic properties has made it important to understand factors that regulate the responsivity of glutamate receptors. In recent years considerable insight has been gained about regulatory sites on NMDA receptors, with the recognition that these receptors are modulated by multiple endogenous and exogenous agents. Less is known about the regulation of responses mediated by AMPA, kainate, ACPD or APB receptors. Desensitization represents a potentially powerful means by which glutamate responses may be regulated. Indeed, two agents closely linked to the physiology of NMDA receptors, glycine and Ca2+, appear to modulate different types of desensitization. In the case of glycine, alteration of a rapid form of desensitization may be important in the role of this amino acid as a necessary cofactor for NMDA receptor activation. Additionally, changes in the affinity of the receptor complex for glycine may underlie the use-dependent decline in NMDA responses under certain conditions. Likewise, Ca2+ is a crucial player in the synaptic and toxic effects mediated by NMDA receptors, and is involved in a slower form of desensitization, in effect helping to regulate its own influx into neurons. The site and mechanism of the Ca2+ regulatory effects remain uncertain with evidence supporting both intracellular and ion channel sites of action. A clear role for Ca(2+)-dependent desensitization in the function of NMDA receptors under physiological conditions has not yet been demonstrated. AMPA receptor desensitization has been an area of intense investigation in recent years. The rapidity and degree of this process, coupled with its apparent rapid recovery, has suggested that desensitization is a key mechanism for the short-term regulation of responses mediated by these receptors. Furthermore, rapid desensitization appears to be one factor determining the time course and efficacy of fast excitatory synaptic transmission mediated by AMPA receptors, highlighting the physiological relevance of the process. The molecular mechanisms underlying desensitization remain uncertain. Traditionally, desensitization, like inactivation of voltage-gated channels, has been thought to represent a conformational change in the ion channel complex (Ochoa et al., 1989). However, it is unknown to what extent desensitization, in particular rapid AMPA receptor desensitization, has mechanistic features in common with inactivation. In voltage-gated channels, conformational changes in the channel protein restrict ion flow through the channel (Stuhmer, 1991).(ABSTRACT TRUNCATED AT 400 WORDS)     

Postsynaptic receptor mechanisms underlying developmental speeding of synaptic transmission

As animals mature the decay of postsynaptic currents become faster at a variety of synapses. This change is thought to contribute to a refinement of motor co-ordination and to an increase in the precision of sensory perception and cognition. At cholinergic neuromuscular synapses and glycinergic and GABAergic inhibitory synapses, the developmental speeding of synaptic currents depends upon switches of receptor subunits and an ensuing acceleration in the kinetics of channel gating. At glutamatergic excitatory synapses, speeding in the decay time of NMDA receptor (NMDAR)-mediated excitatory postsynaptic currents (NMDA-EPSCs) is also dependent on developmental switches in NMDAR subunits. However, developmental speeding in the kinetics of AMPA receptor (AMPAR)-mediated EPSCs (AMPA-EPSCs) is caused by multiple factors. The decay time of AMPA-EPSCs can be shaped by the kinetics of channel gating or desensitization of AMPA receptors, depending upon the speed of transmitter clearance from the synaptic cleft. During postnatal development AMPAR channel gating and desensitization as well as the transmitter clearance speed up in kinetics. Given that the developmental speeding of synaptic currents play critical roles in the maturation of sensory and motor functions, any defect in this mechanism may seriously affect neuronal function.     

A use-dependent tyrosine dephosphorylation of NMDA receptors is independent of ion flux

Tyrosine phosphorylation can upregulate NMDA receptor activity during pathological and physiological alterations of synaptic strength. Here we describe downregulation of recombinant NR1/2A receptors by tyrosine dephosphorylation that requires agonist binding, but is independent of ion flux. The tyrosine residues involved in this new form of NMDA receptor modulation likely form a 'ring' adjacent to the last transmembrane domain. The downregulation was due to a reduction in the number of functional channels, and was blocked by co-expressing a dominant-negative mu2-subunit of the clathrin-adaptor protein AP-2. Our results provide a mechanism by which synaptic NMDA receptors can be modulated in a use-dependent manner even when the postsynaptic membrane is not sufficiently depolarized to relieve channel block by magnesium ions.     

NMDA receptor up-regulates pyroglutamyl peptidase II activity in the rat hippocampus

Ecto-peptidases hydrolyze peptides in the extracellular fluid of the brain. This process is critical for defining the strength of peptidergic communication. A few studies suggest that brain ecto-peptidase activities are regulated by brain function but the extracellular messengers involved are generally unknown. Pyroglutamyl peptidase II (PPII) is specific for thyrotropin releasing hormone (TRH), a tripeptide with multiple homeostatic functions in brain. The purpose of this study was to identify regulators of brain PPII activity. Electrical stimulation (multiple tetani) did not change PPII activity in cortical or hippocampal slices. However, in hippocampal slices, blockade of calcium channels with high magnesium, or of L-type calcium channels (LTCC) or NMDA receptors, decreased PPII activity, while blockade of AMPA or GABA_A receptors did not. Blockade of NMDA receptors did not change PPII mRNA levels but decreased PPII levels. The activity of another ecto-peptidase, aminopeptidase N, was also down regulated by a magnesium blockade, not regulated by NMDA receptor blockade and increased by LTCC blockade. The data show a differential regulation of the activity of ecto-peptidases by that of Ca²⁺ channel and that synaptic activity, through the NMDA receptor, specifically regulates that of pyroglutamyl peptidase II.     

Function of NMDA receptors and persistent sodium channels in a feedback pathway of the electrosensory system

Voltage-dependent amplification of ionotropic glutamatergic excitatory postsynaptic potentials (EPSPs) can, in many vertebrate neurons, be due either to the intrinsic voltage dependence of N-methyl-D-aspartate (NMDA) receptors, or voltage-dependent persistent sodium channels expressed on postsynaptic dendrites or somata. In the electrosensory lateral line lobe (ELL) of the gymnotiform fish Apteronotus leptorhynchus, glutamatergic inputs onto pyramidal cell apical dendrites provide a system where both amplification mechanisms are possible. We have now examined the roles for both NMDA receptors and sodium channels in the control of EPSP amplitude at these synapses. An antibody specific for the A. leptorhynchus NR1 subunit reacted strongly with ELL pyramidal cells and were particularly abundant in the spines of pyramidal cell apical dendrites. We have also shown that NMDA receptors contributed strongly to the late phase of EPSPs evoked by stimulation of the feedback fibers terminating on the apical dendritic spines; further, these EPSPs were voltage dependent. Blockade of NMDA receptors did not, however, eliminate the voltage dependence of these EPSPs. Blockade of somatic sodium channels by local somatic ejection of tetrodotoxin (TTX), or inclusion of QX314 (an intracellular sodium channel blocker) in the recording pipette, reduced the evoked EPSPs and completely eliminated their voltage dependence. We therefore conclude that, in the subthreshold range, persistent sodium currents are the main contributor to voltage-dependent boosting of EPSPs, even when they have a large NMDA receptor component.