Mechanisms of blockade of glutamate receptor ionic channels: Paradox of 9-aminoacridine

9-Aminoacridine and tacrine differ from other channel blockers of NMDA receptors in that their binding prevents the closing of blocked channels and subsequent dissociation of the agonist. Structural determinants of aminoacridine derivatives underlying the blocking mechanism are still unknown. The aim of this study was to elucidate the effects of a dicationic 9-aminoacridine derivative and some other tricyclic compounds on NMDA receptors of rat hippocampal pyramidal neurons. All the compounds under study are voltage-dependent blockers of NMDA channels; their IC₅₀ values recorded at −80 mV vary from 1 to 50 μM. The dicationic derivatives demonstrate the same voltage dependence of the block as the monocationic derivatives. The monoand dicationic tricyclic compounds under study are weak blockers of AMPA receptor channels and differ from adamantane, phenylcyclohexyl and other dicationic derivatives that exhibit greater voltage dependence of the NMDA channel block and are able to induce effective suppression of AMPA channels. We conclude that the mechanisms of action of the tricyclic and dicationic 9-aminoacridine derivatives are different from that of 9-aminoacridine, since these compounds do not prevent closing of the blocked channels. This suggests that the binding site for 9-aminoacridine has specific properties and high selectivity with respect to ligand structure. Original Russian Text ? K.H. Kim, V.E. Gmiro, D.B. Tikhonov, L.G. Magazanik, 2007, published in Biologicheskie Membrany, 2007, Vol. 24, No. 1, pp. 96–104.     

Chronic ethanol exposure attenuates the anti-apoptotic effect of NMDA in cerebellar granule neurons

Ethanol, added to primary cultures of cerebellar granule neurons simultaneously with NMDA, was previously shown to inhibit the anti-apoptotic effect of NMDA. The in vitro anti-apoptotic effect of NMDA is believed to mimic in vivo protection against apoptosis afforded by innervation of developing cerebellar granule neurons by glutamatergic mossy fibers. Therefore, the results suggested that the presence of ethanol in the brain at a critical period of development would promote apoptosis. In the present studies, we examined the effect of chronic ethanol exposure on the anti-apoptotic action of NMDA in cerebellar granule neurons. The neurons were treated with ethanol in vitro for 1-3 days in the absence of NMDA. Even after ethanol was removed from the culture medium, as ascertained by gas chromatography, the protective effect of added NMDA was significantly attenuated. The decreased anti-apoptotic effect of NMDA was associated with a change in the properties of the NMDA receptor, as indicated by a decrease in ligand binding, decreased expression of NMDA receptor subunit proteins, and decreased functional responses including stimulation of increases in intracellular Ca(2+) and induction of brain-derived neurotrophic factor expression. The latter effect may directly underlie the attenuated protective effect of NMDA in these neurons. The results suggest that ethanol exposure during development can have long-lasting effects on neuronal survival. The change in the NMDA receptor caused by chronic ethanol treatment may contribute to the loss of cerebellar granule neurons that is observed in animals and humans exposed to ethanol during gestation.     

Mechanisms of blockade of glutamate receptors channels: the significance for structural and physiological investigations

The mechanism of blocking effect of phenylcyclohexyl derivative, IEM-1925, on ionotropic glutamate receptors of the NMDA and AMPA types has been studied on the rat isolated brain neurons. The whole-cell configuration of patck clanp recording technique was used equilibrium conditions and -80 mV holding potential, the IEM-1925 manifests nonselective action on open channels of both receptors. However, the prominent differences in the mechanism of the blocking effect were revealed. Although IEM-1925 can not enter the closed channels of both types, its molecule are able to leave closed channels of the AMPA but not the NMDA receptors. Hyperpolarization reduces removal of blocker from the open channels of the NMDA receptors. Contrary to that, hyperpolarization facilitates going out of the IEM-1925 to cytozol from both open and closed channels. Evidently, the bloker can pass through the AMPA receptor channels into the cell, and the gating mechanism of these channels is located above the binding site for the blocker. The blocking action of the IEM-1925 on the NMDA and AMPA receptors was compared with its potency to weaken the tremor evoked by subcutaneous injection of arecoline to mice. The observed differences in the mechanisms of action help to explain the ambiguous effects of channel blocking drugs on experimental models of pathological processes.     

Different capacities of various NMDA receptor antagonists to prevent ischemia-induced neurodegeneration in human cultured NT2 neurons

In the present study, human NT2 neurons obtained from embryonic teratocarcinoma (NT2) cells were established as human in-vitro model to investigate the mechanisms associated with hypoxia/ischemia-induced neuronal injury. NT2 neurons express functional NMDA receptors that are of particular significance for hypoxia/ischemia-related neuronal damage. In patch-clamp recordings under normoxic conditions, NMDA (plus 10 microM glycine)-induced inward currents (EC(50)=43.7 microM) were distinctly antagonized by memantine, a blocker of the receptor channel, but only slightly by 5,7-dichlorokynurenic acid (DCKA), a glycine(B) binding site antagonist. Immunohistochemistry demonstrated that the NT2 neurons are mostly GABAergic; they predominantly express the NMDA receptor subunits NR2B and NR2C, and lower levels of NR1 and, particularly, of NR2A. Upon glucose and oxygen deprivation for 3h the loss of cell viability measured directly after 3h was higher than after application of either hypoxia or aglycemia as assessed by propidium iodide flow cytometry. Ischemic conditions significantly reduced the NMDA responses associated with a decrease in EC(50) and decreased mitochondrial membrane potential as detected by JC-1 flow cytometry. Memantine (50 microM) and CGS19755 (a competitive NMDA receptor antagonist; 10 microM) reduced ischemia-induced cell death, in contrast to DCKA (10 microM). In conclusion, in the present human in-vitro model for studying the molecular mechanisms associated with ischemic injury, neuroprotection could be achieved with NMDA receptor antagonists but not with a glycine(B) binding site antagonist. Accordingly, glycine antagonists might not represent an optimal therapeutic strategy for preventing ischemic neuronal damage in contrast to NMDA receptor antagonists like memantine.     

Direct interaction of myosin regulatory light chain with the NMDA receptor

NMDA receptors interact with a variety of intracellular proteins at excitatory synapses. In this paper we show that myosin regulatory light chain (RLC) isolated from mouse brain is a NMDA receptor-interacting protein. Myosin RLC bound directly to the C-termini of both NMDA receptor 1 (NR1) and NMDA receptor 2 (NR2) subunits, rendering the interaction of myosin RLC with NMDA receptors distinct from that of calmodulin which is considered a NR1-interacting protein. Myosin RLC co-localized with NR1 in the dendritic spines of isolated hippocampal neurons, and was co-immunoprecipitated from brain extracts in a complex with NR1, NR2A, NR2B, PSD-95, Adaptor protein-2 and myosin II heavy chain. The C0 region of NR1 was necessary and sufficient for binding myosin RLC. Ca2+/calmodulin, but not calmodulin alone, displaced recombinant myosin RLC from the carboxy tail of NR1 indicating that myosin RLC and Ca2+/calmodulin can compete for a common binding site on NR1 in vitro. Myosin RLC is the only known substrate for myosin regulatory light chain kinase, which has recently been shown to modulate NMDA receptor function in isolated hippocampal neurons. Our results suggest that an additional level of NMDA receptor regulation may be mediated via a direct interaction with a light chain of myosin II. Thus, myosin RLC-NMDA receptor interactions may contribute to the contractile and motile forces that are placed upon NMDA receptor subunits during changes associated with synaptic plasticity and neural morphogenesis.     

The role of NMDA receptors in the slow neuronal degeneration of Parkinson's disease

Summary Parkinson's disease is a disorder, in which neurons of various neuronal systems degenerate. Furthermore, in such degenerating neurons, the cytoskeleton seems to be affected. In this respect, Parkinson's disease resembles Alzheimer's disease. Since it has been shown, that elevated levels of intracellular calcium can disrupt the cytoskeleton and that the stimulation of glutamate (NMDA) receptors can cause high intracellular concentrations of calcium, it has been suggested, that the stimulation of glutamate receptors plays a role in the slow degeneration in Alzheimer's and Parkinson's disease. In case of the degeneration of the dopaminergic nigrostriatal system in Parkinson's disease, neurons that contain calcium binding protein appear to be less vulnerable than the neurons that lack it, suggesting that calcium binding protein might protect these neurons from degeneration by preventing that cytosolic calcium concentrations increase excessively. And, since there is in the nigrostriatal system a glutamatergic afferent pathway (the prefrontonigral projection) and since dopaminergic nigrostriatal neurons contain postsynaptic NMDA receptors, glutamatergic excitation may play a role in the degeneration of the nigrostriatal system in Parkinson's disease. If so, it may be possible to protect the neurodegeneration of these dopaminergic neurons by NMDA receptor antagonists.     

The Arg473Cys-neuroligin-1 mutation modulates NMDA mediated synaptic transmission and receptor distribution in hippocampal neurons

Synapses mediate communication between neurons, thus playing a fundamental role in information processing in the CNS. Neuroligins form a family of heterophilic synaptic cell adhesion molecules, and neuroligin 1 (NL1) has been shown to be involved in the formation of excitatory synapses and have been suggested to associate indirectly with NMDA receptors by common binding to PSD95. A mutation in neuroligin 3 (Arg451Cys-NL3, human sequence numbering) identified in autistic patients is associated with altered spine density and has reduced binding capacity for its presynaptic partner beta-neurexin. Here, we investigated the role of NL1 and the homologous NL1 mutation Arg473Cys-NL1 (R473C-NL1) in excitatory synaptic transmission and NMDA receptor distribution. We demonstrate that R473C-NL1, when expressed in cultured hippocampal neurons, can induce a dramatic increase in NMDA current amplitude and that this change is accompanied by NMDA receptor clustering in the postsynaptic cell.     

Characterization of [3H]MK-801 binding to N-methyl-D-aspartate receptors in cultured rat cerebellar granule neurons and involvement in glutamate-mediated toxicity

The conditions required for growth and survival of cerebellar granule neurons in vitro are known to alter the developmental regulation of NMDA receptor subunit mRNA. In the present report, we have examined the functional and pharmacological characteristics of NMDA receptors on cerebellar granule neurons at 12 days in culture (12 DIC). Under open-channel conditions in extensively washed membranes, [³H]MK-801 labeled a uniform population of sites (K_d = 3.2 ± 0.3 nM) in a saturable manner (B_max = 416 ± 18 fmol/mgl); however, biexponential association and dissociation kinetics indicated the possible existence of at least two NMDA receptor populations that differ in pharmacological properties. The kinetically derived equilibrium dissociation constants for the high- and low-affinity binding components were 0.56 and 771 nM, respectively. The equilibrium competition analysis of MK-801 and other channel-blocking compounds as displacers of [³H]MK-801 revealed the presence of high- and low-affinity binding sites with relative apportionments of 70% and 30%, respectively. The rank-order potency profile of competitor binding at the high-affinity site was (+)-MK-801 > TCP > dextrorphan > dextromethorphan > (+)-ketamine. When tested for the ability to protect 12 DIC cerebellar granule neurons from acute glutamate-induced toxicity, the neuroprotective rank-order potency of these compounds was MK-801 > TCP > dextrorphan > (+)-ketamine > dextromethorphan, which correlated significantly with the high-affinity competition binding profile and thus established the role of NMDA receptors in glutamate toxicity. The findings of these experiments indicate that NMDA receptors on 12 DIC cerebellar granule neurons are a heterogenous population that functionally mediate glutamate-induced neurotoxicity. The heterogenous [³H]MK-801 binding sites may represent NMDA receptor channels composed of different subunits. © 1997 John Wiley & Sons, Inc.     

Enhanced NMDA Receptor-Mediated Modulation of Excitatory Neurotransmission in the Dorsal Vagal Complex of Streptozotocin-Treated,Chronically Hyperglycemic Mice

A variety of metabolic disorders, including complications experienced by diabetic patients, have been linked to altered neural activity in the dorsal vagal complex. This study tested the hypothesis that augmentation of N-Methyl-D-Aspartate (NMDA) receptor-mediated responses in the vagal complex contributes to increased glutamate release in the dorsal motor nucleus of the vagus nerve (DMV) in mice with streptozotocin-induced chronic hyperglycemia (i.e., hyperglycemic mice), a model of type 1 diabetes. Antagonism of NMDA receptors with AP-5 (100 μM) suppressed sEPSC frequency in vagal motor neurons recorded in vitro, confirming that constitutively active NMDA receptors regulate glutamate release in the DMV. There was a greater relative effect of NMDA receptor antagonism in hyperglycemic mice, suggesting that augmented NMDA effects occur in neurons presynaptic to the DMV. Effects of NMDA receptor blockade on mEPSC frequency were equivalent in control and diabetic mice, suggesting that differential effects on glutamate release were due to altered NMDA function in the soma-dendritic membrane of intact afferent neurons. Application of NMDA (300 μM) resulted in greater inward current and current density in NTS neurons recorded from hyperglycemic than control mice, particularly in glutamatergic NTS neurons identified by single-cell RT-PCR for VGLUT2. Overall expression of NR1 protein and message in the dorsal vagal complex were not different between the two groups. Enhanced postsynaptic NMDA responsiveness of glutamatergic NTS neurons is consistent with tonically-increased glutamate release in the DMV in mice with chronic hyperglycemia. Functional augmentation of NMDA-mediated responses may serve as a physiological counter-regulatory mechanism to control pathological disturbances of homeostatic autonomic function in type 1 diabetes.     

Characterization of the gating conformational changes in the felbamate binding site in NMDA channels

The anticonvulsant effect of felbamate (FBM) is ascribable to inhibition of N-methyl-d-aspartate (NMDA) currents. Using electrophysiological studies in rat hippocampal neurons to examine the kinetics of FBM binding to and unbinding from the NMDA channel, we show that FBM modifies NMDA channel gating via a one-to-one binding stoichiometry and has quantitatively the same enhancement effect on NMDA and glycine binding to the NMDA channel. Moreover, the binding rates of FBM to the closed and the open/desensitized NMDA channels are 187.5 and 4.6 x 10(4) M(-1) s(-1), respectively. The unbinding rates of FBM from the closed and the open/desensitized NMDA channels are approximately 6.2 x 10(-2) and approximately 3.1 s(-1), respectively. From the binding and unbinding rate constants, apparent dissociation constants of approximately 300 and approximately 70 microM could be calculated for FBM binding to the closed and the open/desensitized NMDA channels, respectively. The slight (approximately fourfold) difference in FBM binding affinity to the closed and to the open/desensitized NMDA channels thus is composed of much larger differences in the binding and unbinding kinetics (approximately 250- and approximately 60-fold difference, respectively). These findings suggest that the effects of NMDA and glycine binding coalesce or are interrelated before or at the actual activation gate, and FBM binding seems to modulate NMDA channel gating at or after this coalescing point. Moreover, the entrance zone of the FBM binding site very likely undergoes a much larger conformational change along the gating process than that in the binding region(s) of the binding site. In other words, the FBM binding site becomes much more accessible to FBM with NMDA channel activation, although the spatial configurations of the binding ligand(s) for FBM themselves are not altered so much along the gating process.     

Activation kinetics reveal the number of glutamate and glycine binding sites on the N-methyl-D-aspartate receptor.

The activation kinetics of N-methyl-D-aspartate (NMDA) channels in outside-out patches from cultured hippocampal neurons were analyzed to determine the number of glutamate and glycine binding sites per channel. Following rapid steps into high concentrations of glutamate, the activation time course was concentration-independent and limited by transitions between the shut, but fully liganded state and the open state. At lower concentrations, ligand binding was rate-limiting. The resulting sigmoidal activation time course was best fitted by a kinetic model with two glutamate binding sites. Glycine concentration jumps in the continuous presence of glutamate were also best fitted with a two-site model. Agonist and co-agonist binding were better described by an independent, rather than a sequential model. We suggest that the NMDA receptor is at least a tetramer containing four ligand binding subunits, assuming a single binding site per subunit.     

Glutamate Binding to the GluN2B Subunit Controls Surface Trafficking of N-Methyl-D-aspartate (NMDA) Receptors

Trafficking of NMDA receptors to the surface of neurons and to synapses is critical for proper brain function and activity-dependent plasticity. Recent evidence suggests that surface trafficking of other ionotropic glutamate receptors requires ligand binding for exit from the endoplasmic reticulum. Here, we show that glutamate binding to GluN2 is required for trafficking of NMDA receptors to the cell surface. We expressed a panel of GluN2B ligand binding mutants in heterologous cells with GluN1 or in rat cultured neurons and found that surface expression correlates with glutamate efficacy. Such a correlation was found even in the presence of dominant negative dynamin to inhibit endocytosis and surface expression correlated with Golgi localization, indicating differences in forward trafficking. Co-expression of wild type GluN2B did not enhance surface expression of the mutants, suggesting that glutamate must bind to both GluN2 subunits in a tetramer and that surface expression is limited by the least avid of the two glutamate binding sites. Surface trafficking of a constitutively closed cleft GluN2B was indistinguishable from that of wild type, suggesting that glutamate concentrations are typically not limiting for forward trafficking. YFP-GluN2B expressed in hippocampal neurons from GluN2B(-/-) mice rescued synaptic accumulation at similar levels to wild type. Under these conditions, surface synaptic accumulation of YFP-GluN2B mutants also correlated with apparent glutamate affinity. Altogether, these results indicate that glutamate controls forward trafficking of NMDA receptors to the cell surface and to synapses and raise the intriguing idea that NMDA receptors may be functional at intracellular sites.     

NMDA recepter-mediated neuroprotection by essential oils from the rhizomes of Acorus gramineus

Acori graminei Rhizoma (AGR) is shown to exhibit a number of pharmacological actions including sedation and anticonvulsive action. To further characterize its actions in the CNS, the present study evaluated the effects of essential oils (EO) from AGR on the excitotoxic neuronal cell death induced in primary rat cortical cell cultures. EO inhibited the glutamate-induced excitotoxicity in a concentration-dependent manner, with the IC50 of 0.241 mg/ml. EO exerted more potent neuroprotection against the toxicity induced by NMDA (IC50 = 0.139 mg/ml). In contrast, the AMPA-induced toxicity was not inhibited by EO. Receptor-ligand binding studies were performed to investigate the neuroprotective action mechanism. EO dramatically inhibited the specific bindings of a use-dependent NMDA receptorion channel blocker [3H]MK-801, indicating an NMDA receptor antagonist-like action. However, the bindings of [3H]MDL 105,519, a ligand selective for the glycine binding site of NMDA receptor, were not considerably inhibited. These results demonstrated that EO extracted from AGR exhibited neuroprotective effects on cultured cortical neurons through the blockade of NMDA receptor activity, and that the glycine binding site appeared not to be the major site of action.     

Chronic ethanol exposure delays the 'developmental switch' of the NMDA receptor 2A and 2B subunits in cultured cerebellar granule neurons

Chronic ethanol treatment of cultured neurons from various brain areas has been found to increase NMDA receptor function and to alter the levels of some NMDA receptor subunit proteins. Because the cultured neurons are exposed to ethanol during a period when the NMDA receptor is undergoing developmental changes in subunit expression, we wished to determine whether ethanol treatment alters this developmental pattern. We found that 3 days of treatment of cerebellar granule neurons with ethanol, which was previously reported to increase NMDA receptor function, resulted in a delay in the 'developmental switch' of the NR2A and NR2B subunits, i.e. the developmental decrease in NR2B and increase in NR2A protein expression. As a result, the level of NR2B was higher, and that of NR2A was lower, in the ethanol-treated cells than in control cells. Cross-linking experiments showed that the changes in total receptor subunit proteins levels were reflected in cell-surface expressed proteins, indicating changes in the amount of functional receptors. These results were confirmed by a higher potency of glycine at the NMDA receptor in the ethanol-treated cells, as determined by NMDA/glycine-induced increases in intracellular Ca(2+). The results suggest that the mechanism by which ethanol alters NMDA receptor expression in cultured neurons, where receptors are undergoing development, differs from the mechanism of ethanol's effect on NMDA receptors in adult brain. Changes in the proportion of NR2A and NR2B subunits may contribute to effects of ethanol on neuronal development.     

Structural characteristics of ionotropic glutamate receptors revealed by channel blockade

The topography of the channel binding site in glutamate receptors (AMPA and NMDA types of rat brain neurons, receptors of molluscan neurons and insect muscle), and in two subtypes of nicotinic cholinoreceptors (in frog muscle and cat sympathetic ganglion), has been investigated by comparison of the blocking effects of mono- and dicationic derivatives of adamantane and phenylcyclohexyl. The channels studied can be divided into two groups. The first one includes AMPA receptor and glutamate receptors of mollusc and insect, and is characterised by the absence of activity of monocationic drugs and the strong dependence of dicationic once on the internitrogen distance in the drug molecule. The second group includes NMDA receptor and both nicotinic cholinoreceptors. Contrary, here the blocking potency of monocations and dications are practically equal irrespective of molecule length. The data obtained suggest that hydrophobic and nucleophilic components of the binding site are located close to each other in the channels of the NMDA receptor type but are separated by approximately 10 A in the AMPA receptor channel.     

Kinetic analysis of antagonist action at N-methyl-D-aspartic acid receptors. Two binding sites each for glutamate and glycine.

Antagonism of glutamate-receptor responses activated by N-methyl-D-aspartic acid (NMDA) was studied using whole cell voltage clamp recording from mouse dissociated hippocampal neurons cultured for 10-15 d. The kinetics of onset of and recovery from NMDA receptor block during continuous application of NMDA together with either glycine, or L-alanine, were recorded in response to concentration jump application of NMDA- and glycine-binding site directed competitive antagonists, applied with a multibarrel flow pipe under conditions which allowed rapid solution changes around the cell less than 10 ms. Mathematical solutions for both one- and two-equivalent site models for competitive antagonism were determined according to the differential equations outlined by Colquhoun and Hawkes (1977. Proc. R. Soc. Lond. B. 199:231-262). The kinetics of action of D-CPP and D-AP5, NMDA binding site antagonists, and 7Cl-kynurenic acid, a glycine binding site antagonist, were examined for each model. For all these antagonists, the kinetics for the onset of and recovery from antagonism were better fit by the two-equivalent site model, which yielded antagonist microscopic kBoff/kBon values which closely approximated Ki values determined from analysis of equilibrium dose response curves. These results suggest that two molecules of NMDA and two molecules of glycine must bind to the NMDA receptor for activation of ion channel gating.     

Regulation of spinophilin Ser94 phosphorylation in neostriatal neurons involves both DARPP-32-dependent and independent pathways

Spinophilin is a protein phosphatase-1 (PP-1)- and actin-binding protein that is enriched in dendritic spines. Phosphorylation of the actin-binding domain of rat spinophilin at one or more sites by protein kinase A (PKA) inhibits actin binding. Here, we investigated the regulation of mouse spinophilin that contains only a single PKA-site (Ser94) within its actin-binding domain. In vitro phosphorylation of Ser94 resulted in the dissociation of spinophilin from actin filaments. In mouse neostriatal slices, phospho-Ser94 (p-Ser94) was dephosphorylated mainly by PP-1 and also by PP-2A. Activation of dopamine D1 receptors in striatonigral medium spiny neurons, and of adenosine A 2A receptors in striatopallidal medium spiny neurons increased, whereas activation of dopamine D2 receptors in striatopallidal neurons decreased, spinophilin Ser94 phosphorylation. In neostriatal slices from DARPP-32 (dopamine- and cAMP-regulated phosphoprotein of 32 kDa) knockout mice, the effects of D1, D2 and A 2A receptors were largely attenuated. Activation of NMDA receptors decreased Ser94 phosphorylation in a PP-2A-dependent, but DARPP-32-independent, manner. These results suggest that PKA-dependent phosphorylation of spinophilin at Ser94 in both striatonigral and striatopallidal neurons requires synergistic contributions from the PKA and DARPP-32/PP-1 pathways. In addition, PP-2A plays a role in Ser94 dephosphorylation in response to activation of both D2 and NMDA receptors.     

N-methyl-D-aspartate-induced alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA) receptor down-regulation involves interaction of the carboxyl terminus of GluR2/3 with Pick1. Ligand-binding studies using Sindbis vectors carrying AMPA receptor decoys

The dynamics of alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA)-type glutamate receptors, as represented by their exocytosis, endocytosis and cytoskeletal linkage, has often been implicated in N-methyl-d-aspartate (NMDA)-dependent synaptic plasticity. To explore the molecular mechanisms underlying the AMPA receptor dynamics, cultured hippocampal neurons were stimulated with 100 microm NMDA, and the biochemical and pharmacological changes in the ligand binding activity of AMPA receptor complexes and its subunits, GluR1 and GluR2/3, were investigated. The NMDA treatment reduced the total amount of bound [(3)H]AMPA on the surface of the neurons but not in their total membrane fraction. This process was mimicked by a protein kinase C activator, phorbol ester, but blocked by an inhibitor of the same kinase, calphostin C. The NMDA-induced down-regulation of the ligand binding activity was also reflected by the decreased AMPA-triggered channel activity as well as by the cells' reduced immunoreactivity for GluR1. In parallel, the NMDA treatment markedly altered the interaction between the AMPA receptor subunits and their associating molecule(s); the association of PDZ molecules, including Pick1, with GluR2/3 was enhanced in a protein-kinase-C-dependent manner. Viral expression vectors carrying GluR1 and GluR2 C-terminal decoys, both fused to enhanced green fluorescent protein, were transfected into hippocampal neurons to disrupt their interactions. The overexpression of the C-terminal decoy for GluR2 specifically and significantly blocked the NMDA-triggered reduction in [(3)H]AMPA binding, whereas that for GluR1 had no effects. Co-immunoprecipitation using anti-Pick1 antibodies revealed that the overexpressed GluR2 C-terminal decoy indeed prevented Pick1 from interacting with the endogenous GluR2/3. Therefore, these observations suggest that the NMDA-induced down-regulation of the functional AMPA receptors involves the interaction between GluR2/3 subunits and Pick1.     

Kinetics and subunit composition of NMDA receptors in respiratory-related neurons

NMDA receptors are involved in a variety of brainstem functions. The excitatory postsynaptic NMDA currents of pre-Botzinger complex interneurons and hypoglossal motoneurons, which are located in the medulla oblongata, show remarkably fast deactivation kinetics of approximately 30 ms compared with NMDA receptors in other types of neurons. Because structural heterogeneity might be the basis for physiological properties, we examined the expression of six NMDA receptor subunits (NMDAR1, NR2A-2D, and NR3A) plus eight NMDR1 splice variants in pre-Botzinger complex, hypoglossal and, for comparison, neurons from the nucleus of the solitary tract in young rats using single cell multiplex RT-PCR. Expression of NR2A, NR2B, and NR2D was observed in all three cell types while NR3A was much more abundant in pre-Botzinger complex interneurons, which belong to the rhythm generator of respiratory activity. In hypoglossal neurons, the NMDAR1 splice variants NMDAR1-4a and NMDAR1-4b were found. In neurons of the nucleus of the solitary tract, instead of NMDAR1-4b, the NMDAR1-2a splice variant was detected. This differential expression of modulatory splice variants might be the molecular basis for the characteristic functional properties of NMDA receptors, as neurons expressing a special NMDAR1 splice variant at the mRNA level show fast kinetics compared with neurons lacking this splice variant.