The regulatory role of calmodulin in the proteolysis of individual neurofilament proteins by calpain

The in vitro degradation of individual neurofilament proteins by calpain and the effects of calmodulin on this proteolysis were studied. Two major results are reported. First, in the presence of calcium, calmodulin binds to the 200-kD neurofilament protein, but only weakly associates with the 150-kD neurofilament protein. The 70-kD neurofilament protein shows no specific calmodulin-binding. Second, calmodulin inhibits the calpain-mediated degradation of the 200-kD neurofilament protein, but does not alter the hydrolysis of the 150-kD and 70-kD neurofilament proteins. In addition, calmodulin is able to bind to the 200-kD neurofilament protein in the presence of other neurofilament subunits, indicating that calmodulin may play a role in the regulation of the metabolism of the 200-kD neurofilament protein in vivo.     

Calpain product of WT-CRMP2 reduces the amount of surface NR2B NMDA receptor subunit

The brain is particularly vulnerable to ischaemia; however, neurons can become tolerant to ischaemic insult. This tolerance has been shown to involve activation of NMDA receptors, but its mechanisms have not yet been fully elucidated. Using a preconditioning protocol, we show that neurons surviving to a transient NMDA exposure become resistant to the glutamatergic agonist. Using a proteomic approach, we found that alterations of the protein pattern of NMDA-resistant neurons are restricted mainly to the five collapsin response mediator proteins (CRMPs). A sustained increase in calpain activity following NMDA treatment is responsible for the production of cleaved CRMPs. Finally, we provide evidence for the involvement of the cleaved form of WT-CRMP2 in the down-regulation of NR2B. Our data suggests that, beside their role in neuronal morphogenesis, CRMPs may contribute to neuronal plasticity.     

Multiple forms of long-term potentiation and multiple regulatory sites of N-methyl-D-aspartate receptors: Role of the redox site

Long-term potentiation (LTP) is a form of synaptic plasticity thought to be involved in learning and memory. Althrough extensively studied, mainly in the CA1 region of the hippocampus, the mechanisms underlying the induction and expression of LTP are poorly elucidated. This is probably due to the fact that LTP is not a unique process and indeed recent studies have shown that several forms of LTP could be generated depending on the experimental conditions. Furthermore, LTP is generally associated with a long-lasting increase of the synaptic efficacy of AMPA receptors but an increasing number of data also suggested that NMDA receptors could be potentiated as well. NMDA receptor responses are modulated by a large number of extracellular and intracellular events, providing additional possibilities for the generation of LTP. The role of these different modulatory sites of the NMDA receptor and their relation with LTP are reviewed with a particular attention to the redox site which seems to be a selective target to distinguish between AMPA and NMDA-LTP. © 1995 John Wiley & Sons, Inc.     

Developmental and cell-selective variations in N-methyl-D-aspartate receptor degradation by calpain

NMDA receptors play critical roles in synaptic modulation and neurological disorders. In this study, we investigated the developmental changes in NR2 cleavage by NMDA receptor-activated calpain in cultured cortical and hippocampal neurons. Calpain activity increased with development, associated with increased expression of NMDA receptors but not of calpain I. The activation of calpain in immature and mature cortical cultures was inhibited by antagonists of NR1/2B and NR1/2A/2B receptors, whereas the inhibition of NR1/2B receptors did not alter calpain activation in mature hippocampal cultures. The degradation of NR2 subunits by calpain differed with developmental age. NR2A was not a substrate of calpain in mature hippocampal cultures, but was cleaved in immature cortical and hippocampal cultures. NR2B degradation by calpain in cortical cultures decreased with development, but the level of degradation of NR2B in hippocampal cultures did not change. The kinetics of NMDA receptor-gated whole cell currents were also modulated by calpain activation in a manner that varied with developmental stage in vitro. In early (but not later) developmental stages, calpain activation altered the NMDA-evoked current rise time and time constants for both desensitization and deactivation. Our data suggest that the susceptibility of the NMDA receptor to cleavage by calpain varies with neuronal maturity in a manner that may alter its electrophysiological properties.     

Identification of mouse NMDA receptor subunit NR2A C-terminal tyrosine sites phosphorylated by coexpression with v-Src

The protein tyrosine kinase Src is known to regulate NMDA receptors in native neurons. While NR2A, NR2B and NR2D are known to be phosphorylated on tyrosine residues, the exact sites have remained unidentified. Immunoprecipitation of NMDA receptor subunits followed by western blotting was used to analyze the state of tyrosine phosphorylation of recombinant NMDA receptor subunits expressed in HEK293 cells. Using antiphosphotyrosine antibody PY20, we find that on expression in HEK cells, v-Src and Fyn cause detectable tyrosine phosphorylation only of NR2A. Because a stronger signal was produced by the constitutively active v-Src, the general region of v-Src phosphorylation was delimited by expression of a series of truncation mutants of NR2A. Site-directed mutagenesis on candidate sites within the likely region allowed identification of three sites, Y1292, Y1325, and Y1387 that account for a significant fraction of the total PY20 signal. Two of these sites, Y1292 and Y1387, were suggested to control current modulation by Src in previous studies of HEK cells expressing NR1/NR2A. One of these sites, Y1325, has not yet been evaluated for effects on receptor current. A unique tyrosine site, Y1267, was shown not to be a site of detectable phosphorylation, in accordance with its Src-independent regulation of receptor currents.     

Moderate AMPA receptor clustering on the nanoscale can efficiently potentiate synaptic current

The prevailing view at present is that postsynaptic expression of the classical NMDA receptor-dependent long-term potentiation relies on an increase in the numbers of local AMPA receptors (AMPARs). This is thought to parallel an expansion of postsynaptic cell specializations, for instance dendritic spine heads, which accommodate synaptic receptor proteins. However, glutamate released into the synaptic cleft can normally activate only a hotspot of low-affinity AMPARs that occur in the vicinity of the release site. How the enlargement of the AMPAR pool is causally related to the potentiated AMPAR current remains therefore poorly understood. To understand possible scenarios of postsynaptic potentiation, here we explore a detailed Monte Carlo model of the typical small excitatory synapse. Simulations suggest that approximately 50% increase in the synaptic AMPAR current could be provided by expanding the existing AMPAR pool at the expense of 100–200% new AMPARs added at the same packing density. Alternatively, reducing the inter-receptor distances by only 30–35% could achieve a similar level of current potentiation without any changes in the receptor numbers. The NMDA receptor current also appears sensitive to the NMDA receptor crowding. Our observations provide a quantitative framework for understanding the ‘resource-efficient’ ways to enact use-dependent changes in the architecture of central synapses.     

Molecular Dissection of Native Mammalian Forebrain NMDA Receptors Containing the NR1 C2 Exon: Direct Demonstration of NMDA Receptors Comprising NR1, NR2A, and NR2B Subunits Within the Same Complex

Abstract: The subunit compositions of the NR1 C2 exon-containing N -methyl- d -aspartate (NMDA) receptors of adult mammalian forebrain were determined by using a combination of immunoaffinity chromatography and immunoprecipitation studies with NMDA receptor subunit-specific antibodies. NMDA receptors were solubilised by sodium deoxycholate, pH 9, and purified by anti-NR1 C2 antibody affinity chromatography. The purified receptor subpopulation showed immunoreactivity with anti-NR1 C2, anti-NR1 N1, anti-NR1 C2', anti-NR2A, and anti-NR2B NMDA receptor antibodies. The NR1 C2-receptor subpopulation was subjected to immunoprecipitation using anti-NR2B antibodies and the resultant immune pellets analysed by immunoblotting where anti-NR1 C2, anti-NR1 C2', anti-NR2A, and anti-NR2B immunoreactivities were all found. Quantification of the immunoblots showed that 46% of the NR1 C2 immunoreactivity was associated with the NR2B subunit. Of this, 87% (i.e., 40% of total) were NR1 C2/NR2B receptors and 13% (6% of total) were NR1 C2/NR2A/NR2B, thus identifying the triple combination as a minor receptor subset. These results demonstrate directly, for the first time, the coexistence of the NR2A and NR2B subunits in native NMDA receptors. They show the coexistence of two splice forms of the NR1 subunit, i.e., NR1 C2 and NR1 C2', in native receptors and, in addition, they imply an NMDA receptor subpopulation containing four types of NMDA receptor subunit, NR1 C2, NR1 C2', NR2A, and NR2B, which, in accord with molecular size determinations, predicts that the NMDA receptor is at least tetrameric. These results are the first quantitative study of NMDA receptor subtypes and demonstrate molecular heterogeneity for both the NR1 and the NR2 subunits in native forebrain NMDA receptors.     

Lithium protection against glutamate excitotoxicity in rat cerebral cortical neurons: involvement of NMDA receptor inhibition possibly by decreasing NR2B tyrosine phosphorylation

The therapeutic mechanisms of lithium for treating bipolar mood disorder remain poorly understood. Recent studies demonstrate that lithium has neuroprotective actions against a variety of insults. Here, we studied neuroprotective effects of lithium against excitotoxicity in cultured cerebral cortical neurons. Glutamate-induced excitotoxicity in cortical neurons was exclusively mediated by NMDA receptors. Pre-treatment of cortical neurons with LiCl time-dependently suppressed excitotoxicity with maximal protection after 6 days of pre-treatment. Significant protection was observed at the therapeutic and subtherapeutic concentration of 0.2-1.6 mm LiCl with almost complete protection at 1 mM. Neuroprotection was also elicited by valproate, another major mood-stabilizer. The neuroprotective effects of lithium coincided with inhibition of NMDA receptor-mediated calcium influx. Lithium pre-treatment did not alter total protein levels of NR1, NR2A and NR2B subunits of NMDA receptors. However, it did markedly reduce the level of NR2B phosphorylation at Tyr1472 and this was temporally associated with its neuroprotective effect. Because NR2B tyrosine phosphorylation has been positively correlated with NMDA receptor-mediated synaptic activity and excitotoxicity, the suppression of NR2B phosphorylation by lithium is likely to result in the inactivation of NMDA receptors and contributes to neuroprotection against excitotoxicity. This action could also be relevant to its clinical efficacy for bipolar patients.     

NMDA receptor-dependent long-term potentiation comprises a family of temporally overlapping forms of synaptic plasticity that are induced by different patterns of stimulation

N-methyl-d-aspartate receptor (NMDAR)-dependent long-term potentiation (LTP) is extensively studied since it is believed to use the same molecular mechanisms that are required for many forms of learning and memory. Unfortunately, many controversies exist, not least the seemingly simple issue concerning the locus of expression of LTP. Here, we review our recent work and some of the extensive literature on this topic and present new data that collectively suggest that LTP can be explained, during its first few hours, by the coexistence of at least three mechanistically distinct processes that are all triggered by the synaptic activation of NMDARs.     

Transient expression of NMDA receptor subunit NR2B in the developing rat heart

NMDA receptors represent a subtype of the ionotropic glutamate receptor family, comprising three classes of subunits (NR1, NR2A-D, NR3), which exhibit distinct patterns of regional and developmental expression in the CNS. Recently, some NMDA receptor subunits have also been described in adult extraneuronal tissues and keratinocytes. However, their developmental expression patterns are currently unknown. With use of RT-PCR and western blot analysis, the expression of NMDA receptor subunit NR2B was investigated in the developing rat heart. NR2B mRNA and protein were detected in heart tissue of rats from embryonic day 14 until postnatal day 21 but disappeared 10 weeks after birth. In contrast, no NMDA receptor subunit NR1, alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor subunit GluR2, or anchoring postsynaptic density protein-95 could be detected in rat heart at any developmental stage. Confocal microscopy of cultured cardiac myocytes (CMs) from neonatal rats revealed distinct NR2B staining mainly of intracellular structures. However, no functional NMDA receptor could be detected on CMs by whole-cell recordings. In conclusion, high concentrations of NR2B protein can be detected in early rat heart development, but its function still remains elusive.     

NMDA受体通道参与大鼠脊髓背角C纤维诱发电位LTP的表达

以往研究表明,激动NMDA受体是引起海马长时程增强(LTP)的必备条件,而LTP的表达主要与AMPA受体的磷酸化及其受体组装到突触后膜有关.但是,近年来有研究表明NMDA受体通道也参与了LTP的表达.为探讨NMDA受体通道是否参与了脊髓背角C纤维诱发电位LTP的表达,诱导LTP后,分别静脉或脊髓局部给予NMDA受体拮抗剂MK801或APV,观察其作用.发现静脉注射非竞争性NMDA受体MK801(0.1mg/kg)对脊髓LTP无影响,注射0.5mg/kg显著抑制LTP,但是当剂量增高到1.0mg/kg时,抑制作用并未进一步增大.脊髓局部给予MK801也能抑制脊髓背角LTP.为验证上述结果,使用了竞争性NMDA受体拮抗剂APⅤ.结果显示,脊髓局部给予50μmol/LAPⅤ对LTP无影响,100μmol/L对LTP有显著的抑制作用,当浓度升至200μmol/L时,抑制作用并未见进一步增强.因此认为,NMDA受体通道部分地参与了脊髓背角C纤维诱发电位LTP的表达.     

Negative regulation of neurotransmitter release by calpain: a possible involvement of specific SNAP-25 cleavage

Synaptic transmission is conducted by neurotransmitters released from presynaptic nerve terminals by means of Ca2+-dependent exocytosis of synaptic vesicles. Formation of a complex of soluble N-ethylmaleimide-sensitive fusion protein receptor (SNARE) proteins, including vesicle-associated membrane protein-2 (VAMP-2) in the synaptic vesicle membrane, and syntaxin 1 and synaptosomal-associated protein of 25 kDa (SNAP-25) in the plasma membrane, is essential for exocytosis. Ionomycin treatment of cultured rat cerebellar granule cells led to cleavage of SNAP-25, but not syntaxin 1 and VAMP-2, that was dependent on extracellular Ca2+. Cleavage was also induced by N-methyl-D-aspartate (NMDA) treatment, but not by depolarization. The use of various site-specific antibodies to SNAP-25, suggested that the cleavage site was in the N-terminal domain of SNAP-25. Calpain inhibitors abolished the Ca2+-dependent cleavage of SNAP-25 and markedly facilitated Ca2+-dependent glutamate (Glu) release from cerebellar granule cells. These results suggest that calpain may play an important role in the long-lasting regulation of synaptic transmission by suppressing neurotransmitter release, possibly through the proteolytic cleavage of SNAP-25.     

Mapping of the calpain proteolysis products of the junctional foot protein of the skeletal muscle triad junction

Summary The Ca²⁺ activated neutral protease calpain II in a concentration-dependent manner sequentially degrades the Junctional foot protein (JFP) of rabbit skeletal muscle triad junctions in either the triad membrane or as the pure protein. This progression is inhibited by calmodulin. Calpain initially cleaves the 565 kDa JFP monomer into peptides of 160 and 410 kDa, which is subsequently cleaved to 70 and 340 kDa. The 340 kDa peptide is finally cleaved to 140 and 200 kDa or its further products. When the JFP was labeled in the triad membrane with the hydrophobic probe 3-(trifuoromethyl) 3-(m) [¹²⁵I]iodophenyl diazirine and then isolated and proteolysed with calpain II, the [¹²⁵I] was traced from the 565 kDa parent to M _r, 410 kDa and then to 340 kDa, implying that these large fragments contain the majority of the transmembrane segments. A 70-kDa frament was also labeled with the hydrophobic probe, although weakly suggesting an additional transmembrane segment in the middle of the molecule. These transmembrane segments have been predicted to be in the C-terminal region of the JFP. Using an ALOM program, we also predict that transmembrane segments may exist in the 70 kDa fragment. The JFP has eight PEDST sequences; this finding together with the calmodulin inhibition of calpain imply that the JFP is a PEDST-type calpain substrate. Calpain usually cleaves such substrates at or near calmodulin binding sites. Assuming such sites for proteolysis, we propose that the fragments of the JFP correspond to the monomer sequence in the following order from the N-terminus: 160, 70, 140 and 200 kDa. For this model, new calmodulin sequences are predicted to exist near 160 and 225 kDa from the N-terminus. When the intact JFP was labeled with azidoATP, label appeared in the 160 and 140 kDa fragments, which according to the above model contain the GXGXXG sequences postulated as ATP binding sites. This transmembrane segment was predicted by the ALOM program. In addition, calpain and calpastatin activities remained associated with triad component organelles throughout their isolation. These findings and the existence of PEDST sequences suggest that the JFP is normally degraded by calpain in vivo and that degradation is regulated by calpastatin and calmodulin     

NMDA receptor NR2B subunit over-expression increases cerebellar granule cell migratory activity

Glutamate acting on NMDA receptors (NMDARs) is known to influence cerebellar granule cell migration. Subunit composition of NMDARs in granule cells changes characteristically during development: NR2B subunit containing receptors are abundant during migration towards the internal granule cell layer but are gradually replaced by NR2A and/or NR2C subunits once the final position is reached. Cerebellar granule cell migration was investigated using mutant mouse lines either with a deletion of the NR2C gene (NR2C^−/− mice) or expressing NR2B instead of the NR2C subunit (NR2C-2B mice). BrdU-labeling revealed that over-expression of NR2B increased granule cell translocation in vivo , while the lack of NR2C subunit did not have any detectable effects on cell migration. Cellular composition of wild-type and mutant dissociated cerebellar granule cell cultures isolated from 10-day-old cerebella were similar, but NR2C-2B cultures had elevated level of NR2B subunits and intracellular Ca²⁺ imaging revealed higher sensitivity towards the addition of NR2B-selective antagonist in vitro . Time-lapse videomicroscopic observations revealed that average migratory velocity and the proportion of translocating cell bodies were significantly higher in NR2C-2B than in wild-type cultures. Our results provide evidence that NR2B-containing NMDARs can have specialized roles during granule cell migration and can increase migratory speed.     

Activation of calpain by Ca2+: roles of the large subunit N-terminal and domain III-IV linker peptides

The calpains are a family of cysteine proteases with closely related amino acid sequences, but a wide range of Ca(2+) requirements (K(d)). For m-calpain, K(d) is approximately 325microM, for mu-calpain it is approximately 50microM, and for calpain 3 it is not strictly known but may be approximately 0.1microM. On the basis of previous structure determination of m-calpain we postulated that two regions of the calpain large subunits, the N-terminal peptide (residues 1-20) and a domain III-IV linker peptide (residues 514-530 in m-calpain) were important in defining K(d). The mutations Lys10Thr in the N-terminal peptide, and Glu517Pro in the domain linker peptide, reduced K(d) of m-calpain by 30% and 42%, respectively, revealing that these two regions are functionally important. The increased Ca(2+)-sensitivity of these mutants demonstrate that the Lys10-Asp148 salt link and the short beta-sheet interaction involving Glu517 are factors contributing to the high K(d) of m-calpain. Though these two regions are physically remote from the active site and Ca(2+)-binding site, they play significant roles in regulating the response of calpain to Ca(2+). Differences in these interactions in mu-calpain and in calpain 3 are also consistent with their progressively lower K(d) values.     

Stabilization of calpain large subunits by overexpression of truncated calpain small subunit in L8 myoblasts

The objectives were to investigate the function of the small subunit in the calpain system by expression of the autolytic form of this subunit in L8 myoblasts. Rat post-autolysis small subunit (21 kDa) cDNA expression plasmid was transfected into L8 myoblasts and selected by G418 containing medium. The concentrations of cytosolic micro-calpain in transfected cells, SS2 and SS3, were found to be 15.7 and 17.3% higher than that in L8Neo control cells, and the concentrations of cytosolic m-calpain in SS2 and SS3 cells were 23.3 and 16.6% higher than that in control cells (L8Neo). The half-life of micro-calpain in SS3 cells (36.5 h) was longer than that in L8Neo cells (32.4 h), while the half-life of m-calpain in SS3 cells (40.1 h) was longer than that in L8Neo cell (37.5 h). These results indicated that the expression of truncated small subunit increased the stability of micro- and m-calpain large subunits in cytosol.     

Structure of the zinc‐bound amino‐terminal domain of the NMDA receptor NR2B subunit

N‐methyl‐D ‐aspartate (NMDA) receptors belong to the family of ionotropic glutamate receptors (iGluRs) that mediate the majority of fast excitatory synaptic transmission in the mammalian brain. One of the hallmarks for the function of NMDA receptors is that their ion channel activity is allosterically regulated by binding of modulator compounds to the extracellular amino‐terminal domain (ATD) distinct from the L ‐glutamate‐binding domain. The molecular basis for the ATD‐mediated allosteric regulation has been enigmatic because of a complete lack of structural information on NMDA receptor ATDs. Here, we report the crystal structures of ATD from the NR2B NMDA receptor subunit in the zinc‐free and zinc‐bound states. The structures reveal the overall clamshell‐like architecture distinct from the non‐NMDA receptor ATDs and molecular determinants for the zinc‐binding site, ion‐binding sites, and the architecture of the putative phenylethanolamine‐binding site.     

Characterization of the NR1, NR2A, and NR2C receptor proteins.

N-Methyl-D-aspartate (NMDA) receptor subunits were characterized with seven polyclonal antibodies. The antibodies were directed against NR1-A, NR2A-N1, and NR2C-N1, representing N-terminal sequences of the NR1, NR2A, and NR2C subunits, and against NR1-E, NR2A-C1, and NR2C-C1, derived from C-terminal sequences of these subunits. The anti-NR1-D antibody was raised against the putative internal loop of NR1. A size of 118 kDa was found in sodium dodecyl sulfate-polyacrylamide gel electrophoresis for NR1 (from rat brain) detected by anti-NR1-D and -NR1-E, but not anti-NR1-A. With the anti-NR1-A antibody, a 125-kDa protein was discovered that may represent a glutamate receptor not yet characterized. NR2A and NR2C were identified as proteins with sizes of 175 and 140 kDa, respectively. Enzymatic N-deglycosylation generated a 97-kDa protein from NR1, a 105-kDa protein from the 125-kDa protein, a 162-kDa protein from NR2A, and a 127-kDa protein from NR2C. In contrast to the deglycosylation product of the NR2A, the 97- and 127-kDa proteins derived from NR1 and NR2C, respectively, were found significantly smaller than the molecular masses of 103 and 141 kDa, respectively, predicted on the basis of DNA data. These products may represent truncated proteins. The tissue content of the NR1 and NR2A was high in bovine hippocampus and cortex but lower in the cerebellum. In contrast, NR2C was solely found in the cerebellum. The 125-kDa protein was highest in the cerebellum and cortex.