Autophosphorylation-dependent targeting of calcium/ calmodulin-dependent protein kinase II by the NR2B subunit of the N-methyl- D-aspartate receptor

Activation and Thr286 autophosphorylation of calcium/calmodulindependent kinase II (CaMKII) following Ca2+ influx via N-methyl-D-aspartate (NMDA)-type glutamate receptors is essential for hippocampal long term potentiation (LTP), a widely investigated cellular model of learning and memory. Here, we show that NR2B, but not NR2A or NR1, subunits of NMDA receptors are responsible for autophosphorylation-dependent targeting of CaMKII. CaMKII and NMDA receptors colocalize in neuronal dendritic spines, and a CaMKII.NMDA receptor complex can be isolated from brain extracts. Autophosphorylation induces direct high-affinity binding of CaMKII to a 50 amino acid domain in the NR2B cytoplasmic tail; little or no binding is observed to NR2A and NR1 cytoplasmic tails. Specific colocalization of CaMKII with NR2B-containing NMDA receptors in transfected cells depends on receptor activation, Ca2+ influx, and Thr286 autophosphorylation. Translocation of CaMKII because of interaction with the NMDA receptor Ca2+ channel may potentiate kinase activity and provide exquisite spatial and temporal control of postsynaptic substrate phosphorylation.     

High level expression of the NMDAR1 glutamate receptor subunit in electroporated COS cells

The rat N-methyl-D-aspartate (NMDA) glutamate receptor subunit NR1-1a was transiently expressed in COS cells using the technique of electroporation, which was fivefold more efficient than the calcium phosphate precipitation method of transfection. The glycine site antagonist 5,7-[3H]dichlorokynurenic acid labeled a single high-affinity site (KD = 29.6 +/- 6 nM; Bmax = 19.4 +/- 1.6 pmol/mg of protein) in membranes derived from COS cells electroporated with NR1-1a. In contrast to previous reports using transiently transfected human embryonic kidney 293 cells, binding of the noncompetitive antagonist (+)-5-[3H]methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5, 10-imine ([3H]MK-801) was not detected in NR1-1a-transfected COS cells. Although immunofluorescent labeling of electroporated COS cells demonstrated that the NR1-1a protein appears to be associated with the cell membrane, neither NMDA nor glutamate effected an increase in intracellular calcium concentration in fura-2-loaded cells, suggesting that homomeric NR1-1a receptors do not act as functional ligand-gated ion channels. Therefore, COS cells appear to differ from Xenopus oocytes with respect to the transient expression of functional homomeric NR1 receptors. Although expression of NR1-1a is sufficient to reconstitute a glycine binding site with wild-type affinity for antagonists in COS cells, recombinant homomeric NR1-1a receptors do not display properties that are characteristic of native NMDA receptors, such as permeability to Ca2+ and channel occupancy by MK-801, when expressed in this mammalian cell line.     

Brain-derived neurotrophic factor and basic fibroblast growth factor downregulate NMDA receptor function in cerebellar granule cells

Evidence has accumulated to suggest that the NMDA glutamate receptor subtype plays an important role in neuronal degeneration evoked by hypoxia, ischemia, or trauma. Cerebellar granule cells in culture are vulnerable to NMDA-induced neuronal excitotoxicity. In these cells, brain-derived neurotrophic factor (BDNF) and basic fibroblast growth factor (FGF2) prevent the excitotoxic effect of NMDA. However, little is known about the molecular mechanisms underlying the protective properties of these trophic factors. Using cultured rat cerebellar granule cells, we investigated whether BDNF and FGF2 prevent NMDA toxicity by downregulating NMDA receptor function. Western blot and RNase protection analyses were used to determine the expression of the various NMDA receptor subunits (NR1, NR2A, NR2B, and NR2C) after BDNF or FGF2 treatment. FGF2 and BDNF elicited a time-dependent decrease in the expression of NR2A and NR2C subunits. Because NMDA receptor activation leads to increased intracellular Ca2+ concentration ([Ca2+]i), we studied the effect of the BDNF- and FGF2-induced reduction in NR2A and NR2C synthesis on the NMDA-evoked Ca2+ responses by single-cell fura-2 fluorescence ratio imaging. BDNF and FGF2 reduced the NMDA-mediated [Ca2+]i increase with a time dependency that correlates with their ability to decrease NR2A and NR2C subunit expression, suggesting that these trophic factors also induce a functional downregulation of the NMDA receptor. Because sustained [Ca2+]i is believed to be causally related to neuronal injury, we suggest that BDNF and FGF2 may protect cerebellar granule cells against excitotoxicity by altering the NMDA receptor-Ca2+ signaling via a downregulation of NMDA receptor subunit expression.     

Brain spectrin binding to the NMDA receptor is regulated by phosphorylation, calcium and calmodulin

The N-methyl-D-aspartate receptor (NMDA-R) and brain spectrin, a protein that links membrane proteins to the actin cytoskeleton, are major components of post-synaptic densities (PSDs). Since the activity of the NMDA-R channel is dependent on the integrity of actin and leads to calpain-mediated spectrin breakdown, we have investigated whether the actin-binding spectrin may interact directly with NMDA-Rs. Spectrin is reported here to interact selectively in vitro with the C-terminal cytoplasmic domains of the NR1a, NR2A and NR2B subunits of the NMDA-R but not with that of the AMPA receptor GluR1. Spectrin binds at NR2B sites distinct from those of alpha-actinin-2 and members of the PSD95/SAP90 family. The spectrin-NR2B interactions are antagonized by Ca2+ and fyn-mediated NR2B phosphorylation, but not by Ca2+/calmodulin (CaM) or by Ca2+/CaM-dependent protein kinase II-mediated NR2B phosphorylation. The spectrin-NR1 interactions are unaffected by Ca2+ but inhibited by CaM and by protein kinase A- and C-mediated phosphorylations of NR1. Finally, in rat synaptosomes, both spectrin and NR2B are loosened from membranes upon addition of physiological concentrations of calcium ions. The highly regulated linkage of the NMDA-R to spectrin may underlie the morphological changes that occur in neuronal dendrites concurrently with synaptic activity and plasticity.     

Associations between lung function and estimated average exposure to NO2 in eight areas of Switzerland. The SAPALDIA Team. Swiss Study of Air Pollution and Lung Diseases in Adults

Ample experimental evidence indicates that acute beta-amyloid infusion into the nucleus basalis of rats elicits abrupt degeneration of the magnocellular cholinergic neurons projecting to the cerebral cortex. In fact, involvement of a permanent Ca2+ overload, partially via N-methyl-D-aspartate (NMDA) receptors, was proposed as a pivotal mechanism in beta-amyloid-induced neurodegeneration. A definite measure of NMDA receptor-mediated processes and subsequent Ca2+ entry is the induction of Ca2+/calmodulin-activated neuronal nitric oxide synthase (nNOS) in nerve cells. In the present account we therefore assessed activation of nNOS in correlation with cholinergic decline after beta-amyloid(1-42) or beta-amyloid(25-35) infusion into the rat nucleus basalis. The results demonstrate the beta-amyloid conformation-dependent enhancement of cortical nitric oxide synthase (NOS) activity. Furthermore, chronic application of the polyamine site NMDA receptor blocker ifenprodil effectively attenuated beta-amyloid neurotoxicity. We propose that nNOS activation reflects the degree of beta-amyloid-induced excitotoxic injury in a proportional manner. Moreover, Ca2+-mediated processes via NMDA receptors, or direct binding of beta-amyloid to this receptor may be a critical step in the neurotoxic mechanisms in vivo.     

Dynamic changes in NADPH-diaphorase staining reflect activity of nitric oxide synthase: evidence for a dopaminergic regulation of striatal nitric oxide release

In fixed tissue, neuronal NADPH-diaphorase staining results from nitric oxide synthase (NOS) activity. Neuronal NOS only synthesizes nitric oxide once activated by the binding of Ca2+/calmodulin. We show here that neuronal NADPH-diaphorase staining is also dependent on Ca2+/calmodulin, implying that only activated NOS is detected. In addition, in bovine pulmonary endothelial cells, carbachol and bradykinin dramatically and rapidly increase the intensity of NADPH-diaphorase staining. Furthermore, administration of MK801, an NMDA antagonist, decreases neuronal NADPH-diaphorase staining. This suggests that the intensity of the NADPH-diaphorase staining is related to the level of enzyme activation at the moment of tissue fixation. The potential of exploiting this observation to detect cellular activation of NOS is illustrated by the observations that the intensity of NADPH-diaphorase staining in rat striatal neurones is decreased following systemic treatment with the D1-like dopamine receptor antagonist SCH23390, and increased by the D2-like antagonist eticlopride. These results therefore provide strong evidence that the NADPH-diaphorase reaction can be used to monitor NOS activity at a cellular level of resolution, and reveal a dopaminergic regulation of NOS activity in the striatum mediated by D1-like and D2-like dopamine receptors.     

In vitro selection of peptides acting at a new site of NMDA glutamate receptors

Oligomeric N-methyl D-aspartate receptor (NMDAR) in brain is a ligand-gated ion channel that becomes selectively permeable to ions upon binding to ligands. For NMDAR channel, the binding of glutamate and glycine results in opening of the calcium permeable channel. Because the calcium influx mediated by NMDAR is important for synaptic plasticity and excitotoxicity, the function of NMDA receptors has been implicated in both health and disease. Native NMDA receptors are thought to be heteromeric pentamers with a central ion conduction pathway. There are five genes (NR1, 2A, 2B, 2C, and 2D) encoding various subunits that have been cloned, and NR1 is thought to be the essential subunit since it forms a functional channel by itself. To study NMDAR structure and function, we have searched for peptide modulators of NR1 using random peptide bacteriophage libraries. The peptides were identified based on their specific association with a purified receptor fusion protein that contains the putative ligand binding domain. We report the identification of one group of cyclic peptides (Mag-1) with a consensus sequence of CDGLRHMWFC. Using biochemical binding analysis and patch clamp electrophysiological recording, we show that the synthetic Mag-1 peptides cause noncompetitive inhibition of the receptor channel activity.     

Regulation of N-methyl-D-aspartate receptor function by constitutively active protein kinase C

The ability of the constitutively active fragment of protein kinase C (PKM) to modulate N-methyl-D-aspartate (NMDA)-activated currents in cultured mouse hippocampal neurons and acutely isolated CA1 hippocampal neurons from postnatal rats was studied using patch-clamp techniques. The responses of two heterodimeric combinations of recombinant NMDA receptors (NR1a/NR2A and NR1a/NR2B) expressed in human embryonic kidney 293 cells were also examined. Intracellular applications of PKM potentiated NMDA-evoked currents in cultured and isolated CA1 hippocampal neurons. This potentiation was observed in the absence or presence of extracellular Ca2+ and was prevented by the coapplication of the inhibitory peptide protein kinase inhibitor(19-36). Furthermore, the PKM-induced potentiation was not a consequence of a reduction in the sensitivity of the currents to voltage-dependent blockade by extracellular Mg2+. We also found different sensitivities of the responses of recombinant NMDA receptors to the intracellular application of PKM. Some potentiation was observed with the NR1a/NR2A subunits, but none was observed with the NR1a/NR2B combination. Applications of PKM to inside-out patches taken from cultured neurons increased the probability of channel opening without changing single-channel current amplitudes or channel open times. Thus, the activation of protein kinase C is associated with potentiation of NMDA receptor function in hippocampal neurons largely through an increase in the probability of channel opening.     

Rapid Ca2+ entry through Ca2+-permeable AMPA/Kainate channels triggers marked intracellular Ca2+ rises and consequent oxygen radical production

The widespread neuronal injury that results after brief activation of highly Ca2+-permeable NMDA channels may, in large part, reflect mitochondrial Ca2+ overload and the consequent production of injurious oxygen radicals. In contrast, AMPA/kainate receptor activation generally causes slower toxicity, and most studies have not found evidence of comparable oxygen radical production. Subsets of central neurons, composed mainly of GABAergic inhibitory interneurons, express AMPA/kainate channels that are directly permeable to Ca2+ ions. Microfluorometric techniques were performed by using the oxidation-sensitive dye hydroethidine (HEt) to determine whether the relatively rapid Ca2+ flux through AMPA/kainate channels expressed on GABAergic neurons results in oxygen radical production comparable to that triggered by NMDA. Consistent with previous studies, NMDA exposures triggered increases in fluorescence in most cultured cortical neurons, whereas high K+ (50 mM) exposures (causing depolarization-induced Ca2+ influx through voltage-sensitive Ca2+ channels) caused little fluorescence change. In contrast, kainate exposure caused fluorescence increases in a distinct subpopulation of neurons; immunostaining for glutamate decarboxylase revealed the responding neurons to constitute mainly the GABAergic population. The effect of NMDA, kainate, and high K+ exposures on oxygen radical production paralleled the effect of these exposures on intracellular Ca2+ levels when they were monitored with the low-affinity Ca2+-sensitive dye fura-2FF, but not with the high-affinity dye fura-2. Inhibition of mitochondrial electron transport with CN- or rotenone almost completely blocked kainate-triggered oxygen radical production. Furthermore, antioxidants attenuated neuronal injury resulting from brief exposures of NMDA or kainate. Thus, as with NMDA receptor activation, rapid Ca2+ influx through Ca2+-permeable AMPA/kainate channels also may result in mitochondrial Ca2+ overload and consequent injurious oxygen radical production.     

Inhibition of G protein-coupled receptor kinase subtypes by Ca2+/calmodulin

G protein-coupled receptor kinases (GRKs) are implicated in the homologous desensitization of G protein-coupled receptors. Six GRK subtypes have so far been identified, named GRK1 to GRK6. The functional state of the GRKs can be actively regulated in different ways. In particular, it was found that retinal rhodopsin kinase (GRK1), but not the ubiquitous betaARK1 (GRK2), can be inhibited by the photoreceptor-specific Ca2+-binding protein recoverin through direct binding. The present study was aimed to investigate regulation of other GRKs by alternative Ca2+-binding proteins such as calmodulin (CaM). We found that Gbetagamma-activated GRK2 and GRK3 were inhibited by CaM to similar extents (IC50 approximately 2 microM), while a 50-fold more potent inhibitory effect was observed on GRK5 (IC50 = 40 nM). Inhibition by CaM was strictly dependent on Ca2+ and was prevented by the CaM inhibitor CaMBd. Since Gbetagamma, which is a binding target of Ca2+/CaM, is critical for the activation of GRK2 and GRK3, it provides a possible site of interaction between these proteins. However, since GRK5 is Gbetagamma-independent, an alternative mechanism is conceivable. A direct interaction between GRK5 and Ca2+/CaM was revealed using CaM-conjugated Sepharose 4B. This binding does not influence the catalytic activity as demonstrated using the soluble GRK substrate casein. Instead, Ca2+/CaM significantly reduced GRK5 binding to the membrane. The mechanism of GRK5 inhibition appeared to be through direct binding to Ca2+/CaM, resulting in inhibition of membrane association and hence receptor phosphorylation. The present study provides the first evidence for a regulatory effect of Ca2+/CaM on some GRK subtypes, thus expanding the range of different mechanisms regulating the functional states of these kinases.     

Reproductive and thyroid hormone levels in rats following 90-day dietary exposure to PCB 28 (2,4,4''-trichlorobiphenyl) or PCB 77 (3,3''4,4''-tetrachlorobiphenyl)

It has been reported that suramin, an anthelminthic, trypanocidal agent and an inhibitor of P2 receptors, may antagonise N-methyl-D-aspartate (NMDA) subtype of the excitatory amino acid receptors. Both NMDA receptors and P2X subclass of P2 receptors are ligand-gated Ca2+-selective channels and, since the increased influx of Ca2+ into neurons has been linked to neurotoxicity, simultaneous inhibition of P2X and NMDA receptors in vivo by suramin could represent an effective neuroprotective treatment. We have found that suramin inhibited the binding of [3H]CGP 39653 to NMDA receptor binding sites in vitro and reduced the frequency of NMDA channel openings in patch-clamp studies. Suramin (1 mM) had no effect on [3H]kainate binding in vitro. In vivo, intracerebroventricular (I.C.V.) injections of suramin (70 nmol/brain) antagonised convulsive effects of the NMDA agonist (RS)-(tetrazol-5-yl)-glycine (TZG, LY 285265). Suramin, however, did not prevent neurotoxic lesions in the hippocampus caused by I.C.V. administration of TZG. Increasing the dose of suramin resulted in death from severe respiratory depression.     

Calcium channels associated with glutamate receptors

Ionotropic glutamate receptors are nowadays classified into two major categories based on the sensitivity to N-methyl-D-aspartic acid(NMDA) in the mammalian central nervous system. The NMDA channel has much higher permeability to Ca2+ than the non-NMDA channels in most situations, while point mutation could lead to increased permeation of Ca2+ across the non-NMDA channels. Opening of the NMDA channel is under control by a variety of endogenous low molecules, including glycine, polyamines, Mg2+, Zn2+ and H+, in addition to modulation by Ca2+/calmodulin, protein kinases and protein phosphatases. Moreover, the NMDA channel contains a redox site modulatory for the opening. Recent molecular cloning studies have revealed that the NMDA channel is a protein complex consisting of different subunits such as NMDAR1 and NMDAR2 with heterologous expression in the brain. Studies on mice knocked out of a particular gene argue in favor of an essential role of the corresponding subunits in molecular mechanisms underlying neuronal network formation and synaptic plasticity.     

Red flags and religious coping: identifying some religious warning signs among people in crisis

Particulate and soluble (1-3)-beta-glucans are effective in preventing infections by enhancing macrophage and neutrophil functions. However, the mechanisms triggering these enhanced cellular responses are essentially unknown. We recently demonstrated that zymosan, a particulate (1-3)-beta-glucan receptor agonist, caused an influx of Ca2+ in NR8383 rat alveolar macrophages (AMs) and a resulting increase in intracellular Ca2+ (Zhang et al., J. Leukoc. Biol. 62 (1997) 341-348). Since Ca2+ is important in mediating leukocyte responses, we investigated whether other (1-3)-beta-glucans also alter Ca2+ mobilization in AMs. Particulate and soluble (1-3)-beta-glucans derived from Saccharomyces cerevisiae were used in these studies. Like zymosan, particulate (1-3)-beta-glucan (WGPs) caused a concentration-dependent increase in [Ca2+]i, which was inhibited by removal of extracellular Ca2+ and by SKF96365, an inhibitor of receptor-operated Ca2+ channels. When three different soluble (1-3)-beta-glucans, with molecular weights of approximately 11,000, 150,000, and 1,000,000 Da, were tested alone for effects on Ca2+ responses, the low molecular weight (1-3)-beta-glucan produced no effect and the intermediate and high molecular weight (1-3)-beta-glucans caused only a small increase in [Ca2+]i. Interestingly, however, all three soluble (1-3)-beta-glucans could significantly reduce the Ca2+ responses induced by a subsequent exposure to either WGPs or zymosan. These results demonstrate that: 1) particulate (1-3)-beta-glucan activates Ca2+ influx in NR8383 macrophages through receptor-operated Ca2+ channels; 2) soluble (1-3)-beta-glucans do not strongly activate Ca2+ influx in these cells; and 3) soluble (1-3)-beta-glucans significantly inhibit Ca2+ influx induced by WGPs or zymosan. Soluble (1-3)-beta-glucans are likely to prevent Ca2+ influx by competitively binding to the (1-3)-beta-glucan receptors recognizing zymosan and WGPs. The smaller Ca2+ influx induced by soluble (1-3)-beta-glucans may represent only a partial activation of post-receptor signal transduction pathways necessary for inducing Ca2+ influx.     

AMPA and NMDA receptors expressed by differentiating Xenopus spinal neurons

N-methyl--aspartate (NMDA) receptors are often the first ionotropic glutamate receptors expressed at early stages of development and appear to influence neuronal differentiation by mediating Ca2+ influx. Although less well studied, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors also can generate Ca2+ elevations and may have developmental roles. We document the presence of AMPA and NMDA class receptors and the absence of kainate class receptors with whole cell voltage-clamp recordings from Xenopus embryonic spinal neurons differentiated in vitro. Reversal potential measurements indicate that AMPA receptors are permeable to Ca2+ both in differentiated neurons and at the time they first are expressed. The PCa/Pmonocation of 1.9 is close to that of cloned Ca2+-permeable AMPA receptors expressed in heterologous systems. Ca2+ imaging reveals that Ca2+ elevations are elicited by AMPA or NMDA in the absence of Mg2+. The amplitudes and durations of these agonist-induced Ca2+ elevations are similar to those of spontaneous Ca2+ transients known to act as differentiation signals in these cells. Two sources of Ca2+ amplify AMPA- and NMDA-induced Ca2+ elevations. Activation of voltage-gated Ca2+ channels by AMPA- or NMDA-mediated depolarization contributes approximately 15 or 30% of cytosolic Ca2+ elevations, respectively. Activation of either class of receptor produces elevations of Ca2+ that elicit further release of Ca2+ from thapsigargin-sensitive but ryanodine-insensitive stores, contributing an additional approximately 30% of Ca2+ elevations. Voltage-clamp recordings and Ca2+ imaging both show that these spinal neurons express functional AMPA receptors soon after neurite initiation and before expression of NMDA receptors. The Ca2+ permeability of AMPA receptors, their ability to generate significant elevations of [Ca2+]i, and their appearance before synapse formation position them to play roles in neural development. Spontaneous release of agonists from growth cones is detected with glutamate receptors in outside-out patches, suggesting that spinal neurons are early, nonsynaptic sources of glutamate that can influence neuronal differentiation in vivo.     

Experimental evaluation of hepatic functional reserve using 111In colloid for clinical application

Gastric smooth muscle of cats was used to investigate the involvement of protein kinase in vanadate-induced contraction. Vanadate caused a contraction of cat gastric smooth muscle in a dose-dependent manner. Vanadate-induced contraction was totally inhibited by 2 mM EGTA and 1.5 mM LaCl3 and significantly inhibited by 10 microM verapamil and 1 microM nifedipine, suggesting that vanadate-induced contraction is dependent on the extracellular Ca2+ concentration. and the influx of extracellular Ca2+ was mediated through voltage-dependent Ca2+ channel. Both protein kinase C inhibitor and tyrosine kinase inhibitor significantly inhibited the vanadate-induced contraction and the combined inhibitory effect of two protein kinase inhibitors was greater than that of each one. But calmodulin antagonists did not have any influence on the vanadate-induced contraction. On the other hand, both forskolin (1 microM) and sodium nitroprusside (1 microM) significantly inhibited vanadate-induced contraction. Therefore, these results suggest that both protein kinase C and tyrosine kinase are involved in the vanadate-induced contraction which required the influx of extracellular Ca2+ in cat gastric smooth muscle, and that the contractile mechanism of vanadate may be different from that of agonist binding to its specific receptor.     

Polyol pathway, 2,3-diphosphoglycerate in erythrocytes and diabetic neuropathy in rats

Despite substantial data on radioligand binding to the sigma receptor, neither the physiologic function nor the intracellular mechanism of this receptor is known. In this study, we examined the effect of sigma ligands on Ca++ influx induced by N-methyl-D-aspartate (NMDA) in single primary cultured rat frontal cortical neurons with fluorescence video microscopy. All sigma ligands tested reduced the NMDA-induced increase in intracellular Ca++ concentration ([Ca++]i) in a dose-dependent manner with IC50 values in the low micromolar range. Inhibition by haloperidol and (+)-N-cyclopropylmethyl-N-methyl-1,4-diphenyl-1-ethyl-but-3-en-1-ylam ine hydrochloride (JO1784) was noncompetitive; but, exogenous glycine (100 microM) did not alter their IC50 values. In addition, haloperidol (1 microM) enhanced Mg+(+)-mediated inhibition of the NMDA-induced [Ca++]i increase (IC50 = 0.45 +/- 0.01 mM vs. an IC50 = 0.98 +/- 0.06 mM for Mg++ alone). Selective sigma receptor ligands (JO1784, (+)-pentazocine) caused a greater reduction of the sustained phase of the Ca++ response to NMDA, whereas haloperidol and DTG reduced both the initial and sustained phase of the response to a similar degree. The rank order of potencies for inhibition of both the sustained Ca++ response phase and (+)-[3H]SKF-10047 binding (Roman et al., J. Pharm. Pharmacol. 42: 439-440, 1989) were similar. These findings suggest that sigma 1 ligands indirectly modulate NMDA receptor complex function through sigma 1 receptors and that sigma ligands facilitate the desensitization of the Ca++ response to NMDA.     

Regulation of heterologously expressed transient receptor potential-like channels by calcium ions

The Drosophila melanogaster gene product TRPL (transient receptor potential-like) is a Ca2+-permeable cation channel that contributes to the light-induced Ca2+ entry in Drosophila photoreceptors and bears homology to several recently cloned mammalian channels. Intracellular Ca2+ has been implicated to stimulate TRPL channels. This constitutes a potentially dangerous mechanism that may lead to Ca2+ overload. Therefore, we studied whether TRPL channels, like other Ca2+-permeable channels, are inhibited by intracellular Ca2+ concentrations in the micromolar range and whether this effect is mediated by calmodulin. In Sf9 cells expressing the TRPL gene along with histamine H1 receptors after infection with baculoviruses containing the corresponding complementary DNA, histamine-induced TRPL currents were inhibited by intracellular Ca2+ with an IC50 of 2.3 microM. Moreover, TRPL currents were reversibly attenuated by a preceding hyperpolarization. This attenuation reflected the action of an increased Ca2+ influx, since it was abolished in the absence of extracellular Ca2+ and enhanced by raising extracellular Ca2+ to 20 mM. Finally, the activity of TRPL channels in inside-out patches was reversibly inhibited by raising the Ca2+ concentration on the cytosolic side of the patches to 10-50 microM. Addition of calmodulin or the calmodulin inhibitor calmidazolium did not modify the inhibition of the TRPL by Ca2+. We conclude that high intracellular Ca2+ concentrations inhibit the TRPL, but no evidence was found for the requirement of calmodulin. This mechanism makes Ca2+ influx through the TRPL self-limiting. Furthermore, the TRPL may allow one to study the structural requirements for channel regulation by Ca2+.     

An acidic amino acid in the N-methyl-D-aspartate receptor that is important for spermine stimulation

The polyamine spermine has multiple effects on N-methyl-D-aspartate (NMDA) receptors, including "glycine-independent" stimulation, which is seen in the presence of saturating concentrations of glycine; "glycine-dependent" stimulation, which is due to an increase in the affinity of the receptor for glycine; and voltage-dependent block. These effects may involve three separate polyamine binding sites on the receptor. To identify amino acid residues that are important for spermine binding, we used site-directed mutagenesis to alter amino acids in and around a region of the NR1 subunit of the NMDA receptor that shows homology with PotD, a polyamine binding protein from Escherichia coli. Mutated subunits, expressed in heteromeric and homomeric NMDA receptors, were studied by voltage-clamp recording in Xenopus oocytes. Mutation of two acidic residues (E339-E342) to neutral amino acids reduced or abolished glycine-independent stimulation by spermine without affecting glycine-dependent stimulation or voltage-dependent block by spermine. Mutation of these residues also had modest effects on sensitivity to protons and to ifenprodil but did not alter sensitivity to glutamate and glycine or to voltage-dependent block by Mg2+. Residue E342 in NR1 appears to be critical for glycine-independent spermine stimulation. Mutations at equivalent positions in NR2A(E352Q) or NR2B(E353Q) had no effect on sensitivity to spermine, pH, or ifenprodil. Residue E342 in NR1 may form part of a discrete spermine binding site on the NMDA receptor or be involved in the mechanism of modulation by polyamines. This residue may also be involved in modulation by protons and ifenprodil.     

Functional change of NMDA receptors related to enhancement of susceptibility to neurotoxicity in the developing pontine nucleus

The developing neurons have been reported to be extremely susceptible to toxicity of NMDA during a restricted developmental period. Pontosubicular neuronal necrosis is a typical type of perinatal human brain lesion and often coexists with other forms of cerebral hypoxic and ischemic injuries. To determine whether functional changes of NMDA receptors related to the susceptibility to NMDA toxicity are involved in developing neurons in the pontine nucleus, we have examined the lesion produced by in vivo direct injection of NMDA into the pontine nucleus of rats at postnatal days 1-30, recorded NMDA-induced whole-cell currents from neurons in the pontine nucleus in the developing rat brainstem slices, and performed in situ hybridization for NMDA receptor subunit mRNAs in the pontine nucleus. The susceptibility to NMDA neurotoxicity peaked near postnatal day 15, and the NMDA-induced currents showed prominent reduction of the voltage-dependent block by Mg2+ near postnatal day 15. The pontine nucleus near postnatal day 15 showed distinct expression of the NMDA receptor subunit NR2C mRNA. These results suggest that the susceptibility to NMDA neurotoxicity that is enhanced in the rat pontine nucleus near postnatal day 15 is mediated by the NMDA receptor channels that are relatively insensitive to Mg2+ and that the reduction in the sensitivity of NMDA receptors to Mg2+ correlates with the expression of the NR2C. We present the possibility that functional changes in the NMDA receptor channels play a crucial role in the occurrence of developmentally specific neuronal injury.     

State-dependent NMDA receptor antagonism by Ro 8-4304, a novel NR2B selective, non-competitive, voltage-independent antagonist

1. Subunit-selective blockade of N-methyl-D-aspartate (NMDA) receptors provides a potentially attractive strategy for neuroprotection in the absence of undesirable side effects. Here, we describe a novel NR2B-selective NMDA antagonist, 4-?3-[4-(4-fluoro-phenyl)-3,6-dihydro-2H-pyridin-1-yl]-2-hydroxy-propoxy ?-benzamide (Ro 8-4304), which exhibits >100 fold higher affinity for recombinant NR1(001)/NR2B than NR1(001)/NR2A receptors. 2. Ro 8-4304 is a voltage-independent, non-competitive antagonist of NMDA receptors in rat cultured cortical neurones and exhibits a state-dependent mode of action similar to that described for ifenprodil. 3. The apparent affinity of Ro 8-4304 for the NMDA receptor increased in an NMDA concentration-dependent manner so that Ro 8-4304 inhibited 10 and 100 microM NMDA responses with IC50s of 2.3 and 0.36 microM, respectively. Currents elicited by 1 microM NMDA were slightly potentiated in the presence of 10 microM Ro 8-4304, and Ro 8-4304 binding slowed the rate of glutamate dissociation from NMDA receptors. 4. These results were predicted by a reaction scheme in which Ro 8-4304 exhibits a 14 and 23 fold higher affinity for the activated and desensitized states of the NMDA receptor, respectively, relative to the agonist-unbound resting state. Additionally, Ro 8-4304 binding resulted in a 3 4 fold increase in receptor affinity for glutamate site agonists. 5. Surprisingly, whilst exhibiting a similar affinity for NR2B-containing NMDA receptors as ifenprodil, Ro 8-4304 exhibited markedly faster kinetics of binding and unbinding to the NMDA receptor. This spectrum of kinetic behaviour reveals a further important feature of this emerging class of NR2B-selective compounds.