Developmental characteristics of AMPA receptors in chick lumbar motoneurons

Ca2+ fluxes through ionotropic glutamate receptors regulate a variety of developmental processes, including neurite outgrowth and naturally occurring cell death. In the CNS, NMDA receptors were originally thought to be the sole source of Ca2+ influx through glutamate receptors; however, AMPA receptors also allow a significant influx of Ca2+ ions. The Ca2+ permeability of AMPA receptors is regulated by the insertion of one or more edited GluR2 subunits. In this study, we tested the possibility that changes in GluR2 expression regulate the Ca2+ permeability of AMPA receptors during a critical period of neuronal development in chick lumbar motoneurons. GluR2 expression is absent between embryonic day (E) 5 and E7, but increases significantly by E8 in the chick ventral spinal cord. Increased GluR2 protein expression is correlated with parallel changes in GluR2 mRNA in the motoneuron pool. Electrophysiological recordings of kainate-evoked currents indicate a significant reduction in the Ca2(+)-permeability of AMPA receptors between E6 and E11. Kainate-evoked currents were sensitive to the AMPA receptor blocker GYKI 52466. Application of AMPA or kainate generates a significant increase in the intracellular Ca2+ concentration in E6 spinal motoneurons, but generates a small response in older neurons. Changes in the Ca(2+)-permeability of AMPA receptors are not mediated by age-dependent changes in the editing pattern of GluR2 subunits. These findings raise the possibility that Ca2+ influx through Ca(2+)-permeable AMPA receptors plays an important role during early embryonic development in chick spinal motoneurons.     

Activation of D1 dopamine receptors increases surface expression of AMPA receptors and facilitates their synaptic incorporation in cultured hippocampal neurons

Considerable evidence indicates that neuroadaptations leading to addiction involve the same cellular processes that enable learning and memory, such as long-term potentiation (LTP), and that psychostimulants influence LTP through dopamine (DA)-dependent mechanisms. In hippocampal CA1 pyramidal neurons, LTP involves insertion of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors into excitatory synapses. We used dissociated cultures to test the hypothesis that D1 family DA receptors influence synaptic plasticity in hippocampal neurons by modulating AMPA receptor trafficking. Brief exposure (5 min) to a D1 agonist increased surface expression of glutamate receptor (GluR)1-containing AMPA receptors by increasing their rate of externalization at extrasynaptic sites. This required the secretory pathway but not protein synthesis, and was mediated mainly by protein kinase A (PKA) with a smaller contribution from Ca2+-calmodulin-dependent protein kinase II (CaMKII). Prior D1 receptor stimulation facilitated synaptic insertion of GluR1 in response to subsequent stimulation of synaptic NMDA receptors with glycine. Our results support a model for synaptic GluR1 incorporation in which PKA is required for initial insertion into the extrasynaptic membrane whereas CaMKII mediates translocation into the synapse. By increasing the size of the extrasynaptic GluR1 pool, D1 receptors may promote LTP. Psychostimulants may usurp this mechanism, leading to inappropriate plasticity that contributes to addiction-related behaviors.     

PICK1 is a calcium-sensor for NMDA-induced AMPA receptor trafficking

Regulation of AMPA receptor (AMPAR) trafficking results in changes in receptor number at the postsynaptic membrane, and hence modifications in synaptic strength, which are proposed to underlie learning and memory. NMDA receptor-mediated postsynaptic Ca2+ influx enhances AMPAR internalisation, but the molecular mechanisms that trigger such trafficking are not well understood. We investigated whether AMPAR-associated protein-protein interactions known to regulate receptor surface expression may be directly regulated by Ca2+. PICK1 binds the AMPAR GluR2 subunit and is involved in AMPAR internalisation and LTD. We show that PICK1 is a Ca2+-binding protein, and that PICK1-GluR2 interactions are enhanced by the presence of 15 muM Ca2+. Deletion of an N-terminal acidic domain in PICK1 reduces its ability to bind Ca2+, and renders the GluR2-PICK1 interaction insensitive to Ca2+. Overexpression of this Ca2+-insensitive mutant occludes NMDA-induced AMPAR internalisation in hippocampal neurons. This work reveals a novel postsynaptic Ca2+-binding protein that provides a direct mechanistic link between NMDAR-mediated Ca2+ influx and AMPAR endocytosis.     

NSF binds calcium to regulate its interaction with AMPA receptor subunit GluR2

N-ethylmaleimide-sensitive fusion protein (NSF) is essential for numerous Ca(2+)-triggered vesicle trafficking events. It functions as a molecular chaperone to regulate trafficking protein complexes such as the soluble NSF attachment protein (SNAP) receptor complex and the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR)-protein interacting with C-kinase (PICK1) complex. AMPAR trafficking is fundamental to processes of synaptic plasticity, which may underlie learning and memory. Changes in synaptic strength brought about by AMPAR trafficking are triggered by a post-synaptic influx of Ca(2+), which may have numerous molecular targets including PICK1. NSF binds AMPAR subunit glutamate receptor subunit 2 (GluR2) and functions to maintain receptors at the synapse. In this study, it was showed that NSF is a Ca(2+)-binding protein and that GluR2-NSF interactions are inhibited by the presence of 15 mumol/L Ca(2+). NSF Ca(2+)-binding is reciprocally inhibited by the presence of GluR2 C-terminus. Mutant of NSF that binds Ca(2+) with reduced affinity and binds GluR2 with reduced sensitivity to Ca(2+) was identied. In addition, the interaction of betaSNAP with PICK1 is sensitive to Ca(2+). This study demonstrates that the GluR2-NSF-betaSNAP-PICK1 complex is regulated directly by Ca(2+), allowing for the transduction of Ca(2+) signals into concerted alterations in protein-protein interactions to bring about changes in AMPAR trafficking during synaptic plasticity.     

Brain-derived neurotrophic factor regulates AMPA receptor trafficking to post-synaptic densities via IP3R and TRPC calcium signaling

The change in the number of post-synaptic alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)-type glutamatergic receptors (AMPARs) by neuronal activity is recognized as a molecular basis of synaptic plasticity. Here, we show that Ca(2+) transients evoked by brain-derived neurotrophic factor (BDNF) induce translocation of a subunit of AMPAR, GluR1, but not NMDAR, to the post-synaptic membrane in cultured cortical pyramidal neurons. Among BDNF-induced Ca(2+) transients, that dependent on IP3R was fully required, while store-operated calcium influx through the non-selective cation channel TRPC (transient receptor potential canonical) was partially required for the GluR1 up-regulation, suggesting that spatial and temporal calcium signaling regulate translocation of GluR1 to the polarized membrane domain.     

Miniature neurotransmission stabilizes synaptic function via tonic suppression of local dendritic protein synthesis

Activity deprivation in neurons induces a slow compensatory scaling up of synaptic strength, reflecting a homeostatic mechanism for stabilizing neuronal activity. Prior studies have focused on the loss of action potential (AP) driven neurotransmission in synaptic homeostasis. Here, we show that the miniature synaptic transmission that persists during AP blockade profoundly shapes the time course and mechanism of homeostatic scaling. A brief blockade of NMDA receptor (NMDAR) mediated miniature synaptic events ("minis") rapidly scales up synaptic strength, over an order of magnitude faster than with AP blockade alone. The rapid scaling induced by NMDAR mini blockade is mediated by increased synaptic expression of surface GluR1 and the transient incorporation of Ca2+-permeable AMPA receptors at synapses; both of these changes are implemented locally within dendrites and require dendritic protein synthesis. These results indicate that NMDAR signaling during miniature synaptic transmission serves to stabilize synaptic function through active suppression of dendritic protein synthesis.     

Calcium-permeable AMPA receptor plasticity is mediated by subunit-specific interactions with PICK1 and NSF

A recently described form of synaptic plasticity results in dynamic changes in the calcium permeability of synaptic AMPA receptors. Since the AMPA receptor GluR2 subunit confers calcium permeability, this plasticity is thought to occur through the dynamic exchange of synaptic GluR2-lacking and GluR2-containing receptors. To investigate the molecular mechanisms underlying this calcium-permeable AMPA receptor plasticity (CARP), we examined whether AMPA receptor exchange was mediated by subunit-specific protein-protein interactions. We found that two GluR2-interacting proteins, the PDZ domain-containing Protein interacting with C kinase (PICK1) and N-ethylmaleimide sensitive fusion protein (NSF), are specifically required for CARP. Furthermore, PICK1, but not NSF, regulates the formation of extrasynaptic plasma membrane pools of GluR2-containing receptors that may be laterally mobilized into synapses during CARP. These results demonstrate that PICK1 and NSF dynamically regulate the synaptic delivery of GluR2-containing receptors during CARP and thus regulate the calcium permeability of AMPA receptors at excitatory synapses.     

Phosphorylation of AMPA receptors is required for sensory deprivation-induced homeostatic synaptic plasticity

Sensory experience, and the lack thereof, can alter the function of excitatory synapses in the primary sensory cortices. Recent evidence suggests that changes in sensory experience can regulate the synaptic level of Ca(2+)-permeable AMPA receptors (CP-AMPARs). However, the molecular mechanisms underlying such a process have not been determined. We found that binocular visual deprivation, which is a well-established in vivo model to produce multiplicative synaptic scaling in visual cortex of juvenile rodents, is accompanied by an increase in the phosphorylation of AMPAR GluR1 (or GluA1) subunit at the serine 845 (S845) site and the appearance of CP-AMPARs at synapses. To address the role of GluR1-S845 in visual deprivation-induced homeostatic synaptic plasticity, we used mice lacking key phosphorylation sites on the GluR1 subunit. We found that mice specifically lacking the GluR1-S845 site (GluR1-S845A mutants), which is a substrate of cAMP-dependent kinase (PKA), show abnormal basal excitatory synaptic transmission and lack visual deprivation-induced homeostatic synaptic plasticity. We also found evidence that increasing GluR1-S845 phosphorylation alone is not sufficient to produce normal multiplicative synaptic scaling. Our study provides concrete evidence that a GluR1 dependent mechanism, especially S845 phosphorylation, is a necessary pre-requisite step for in vivo homeostatic synaptic plasticity.     

Reinsertion or degradation of AMPA receptors determined by activity-dependent endocytic sorting

Both acute and chronic changes in AMPA receptor (AMPAR) localization are critical for synaptic formation, maturation, and plasticity. Here I report that AMPARs are differentially sorted between recycling and degradative pathways following endocytosis. AMPAR sorting occurs in early endosomes and is regulated by synaptic activity and activation of AMPA and NMDA receptors. AMPAR intemalization triggered by NMDAR activation is Ca2+-dependent, requires protein phosphatases, and is followed by rapid membrane reinsertion. Furthermore, NMDAR-mediated AMPAR trafficking is regulated by PKA and accompanied by dephosphorylation and rephosphorylation of GluR1 subunits at a PKA site. In contrast, activation of AMPARs without NMDAR activation targets AMPARs to late endosomes and lysosomes, independent of Ca2+, protein phosphatases, or PKA. These results demonstrate that activity regulates AMPAR endocytic sorting, providing a potential mechanistic link between rapid and chronic changes in synaptic strength.     

Ca2+ Permeable AMPA Receptor Induced Long-Term Potentiation Requires PI3/MAP Kinases but Not Ca/CaM-Dependent Kinase II

Ca²⁺ influx via GluR2-lacking Ca²⁺-permeable AMPA glutamate receptors (CP-AMPARs) can trigger changes in synaptic efficacy in both interneurons and principle neurons, but the underlying mechanisms remain unknown. We took advantage of genetically altered mice with no or reduced GluR2, thus allowing the expression of synaptic CP-AMPARs, to investigate the molecular signaling process during CP-AMPAR-induced synaptic plasticity at CA1 synapses in the hippocampus. Utilizing electrophysiological techniques, we demonstrated that these receptors were capable of inducing numerous forms of long-term potentiation (referred to as CP-AMPAR dependent LTP) through a number of different induction protocols, including high-frequency stimulation (HFS) and theta-burst stimulation (TBS). This included a previously undemonstrated form of protein-synthesis dependent late-LTP (L-LTP) at CA1 synapses that is NMDA-receptor independent. This form of plasticity was completely blocked by the selective CP-AMPAR inhibitor IEM-1460, and found to be dependent on postsynaptic Ca²⁺ ions through calcium chelator (BAPTA) studies. Surprisingly, Ca/CaM-dependent kinase II (CaMKII), the key protein kinase that is indispensable for NMDA-receptor dependent LTP at CA1 synapses appeared to be not required for the induction of CP-AMPAR dependent LTP due to the lack of effect of two separate pharmacological inhibitors (KN-62 and staurosporine) on this form of potentiation. Both KN-62 and staurosporine strongly inhibited NMDA-receptor dependent LTP in control studies. In contrast, inhibitors for PI3-kinase ({"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 and wortmannin) or the MAPK cascade (PD98059 and U0126) significantly attenuated this CP-AMPAR-dependent LTP. Similarly, postsynaptic infusion of tetanus toxin (TeTx) light chain, an inhibitor of exocytosis, also had a significant inhibitory effect on this form of LTP. These results suggest that distinct synaptic signaling underlies GluR2-lacking CP-AMPAR-dependent LTP, and reinforces the recent notions that CP-AMPARs are important facilitators of synaptic plasticity in the brain.     

The phosphorylation state of GluR1 subunits determines the susceptibility of AMPA receptors to calpain cleavage

The alpha-Amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid receptor (AMPAR) is an ionotropic glutamate receptor that governs most of excitatory synaptic transmission in neurons. In vitro biochemical assay has shown that calpain, a Ca2+-activated protease, can cleave AMPAR GluR1 subunits. Our physiological study found that calpain, which was activated by prolonged stimulation of the N-methyl-D-aspartate receptor (100 microM, 10 min), caused a substantial suppression of AMPAR currents in cortical neurons. Since the phosphorylation sites of GluR1 by several protein kinases are located in close proximity to the calpain cleavage sites, we investigated the effect of phosphorylation on the susceptibility of GluR1 to calpain cleavage. Interestingly, we found that the calpain regulation of AMPAR currents was diminished by inhibition of Ca2+/calmodulin-dependent protein kinase II (CaMKII) but was augmented by inhibition of protein phosphatase 1/2A (PP1/2A). In agreement with this, in vitro assay showed that the calpain-induced proteolytic cleavage of GluR1 C-terminal fusion protein was strongly potentiated by adding the purified active CaMKII, and GluR1 phosphorylated at Ser831 by CaMKII is much more sensitive to calpain cleavage. Taken together, our data suggest that calpain activation suppresses AMPA receptor currents via proteolytic cleavage of GluR1 subunits, and the susceptibility of AMPARs to calpain cleavage is determined by the phosphorylation state of GluR1 subunits, which is mediated by CaMKII-PP1/2A activity.     

Subunit-specific rules governing AMPA receptor trafficking to synapses in hippocampal pyramidal neurons

AMPA-type glutamate receptors (AMPA-Rs) mediate a majority of excitatory synaptic transmission in the brain. In hippocampus, most AMPA-Rs are hetero-oligomers composed of GluR1/GluR2 or GluR2/GluR3 subunits. Here we show that these AMPA-R forms display different synaptic delivery mechanisms. GluR1/GluR2 receptors are added to synapses during plasticity; this requires interactions between GluR1 and group I PDZ domain proteins. In contrast, GluR2/GluR3 receptors replace existing synaptic receptors continuously; this occurs only at synapses that already have AMPA-Rs and requires interactions by GluR2 with NSF and group II PDZ domain proteins. The combination of regulated addition and continuous replacement of synaptic receptors can stabilize long-term changes in synaptic efficacy and may serve as a general model for how surface receptor number is established and maintained.     

PICK1-mediated Glutamate Receptor Subunit 2 (GluR2) Trafficking Contributes to Cell Death in Oxygen/Glucose-deprived Hippocampal Neurons

Oxygen and glucose deprivation (OGD) induces delayed cell death in hippocampal CA1 neurons via Ca²⁺/Zn²⁺-permeable, GluR2-lacking AMPA receptors (AMPARs). Following OGD, synaptic AMPAR currents in hippocampal neurons show marked inward rectification and increased sensitivity to channel blockers selective for GluR2-lacking AMPARs. This occurs via two mechanisms: a delayed down-regulation of GluR2 mRNA expression and a rapid internalization of GluR2-containing AMPARs during the OGD insult, which are replaced by GluR2-lacking receptors. The mechanisms that underlie this rapid change in subunit composition are unknown. Here, we demonstrate that this trafficking event shares features in common with events that mediate long term depression and long term potentiation and is initiated by the activation of N-methyl-d-aspartic acid receptors. Using biochemical and electrophysiological approaches, we show that peptides that interfere with PICK1 PDZ domain interactions block the OGD-induced switch in subunit composition, implicating PICK1 in restricting GluR2 from synapses during OGD. Furthermore, we show that GluR2-lacking AMPARs that arise at synapses during OGD as a result of PICK1 PDZ interactions are involved in OGD-induced delayed cell death. This work demonstrates that PICK1 plays a crucial role in the response to OGD that results in altered synaptic transmission and neuronal death and has implications for our understanding of the molecular mechanisms that underlie cell death during stroke.Oxygen and glucose deprivation (OGD)³ associated with transient global ischemia induces delayed cell death, particularly in hippocampal CA1 pyramidal cells (1–3), a phenomenon that involves Ca²⁺/Zn²⁺-permeable, GluR2-lacking AMPARs (4). AMPARs are heteromeric complexes of subunits GluR1–4 (5), and most AMPARs in the hippocampus contain GluR2, which renders them calcium-impermeable and results in a marked inward rectification in their current-voltage relationship (6–8). Ischemia induces a delayed down-regulation of GluR2 mRNA and protein expression (4, 9–11), resulting in enhanced AMPAR-mediated Ca²⁺ and Zn²⁺ influx into CA1 neurons (10, 12). In these neurons, AMPAR-mediated postsynaptic currents (EPSCs) show marked inward rectification 1–2 days following ischemia and increased sensitivity to 1-naphthyl acetyl spermine (NASPM), a channel blocker selective for GluR2-lacking AMPARs (13–16). Blockade of these channels at 9–40 h following ischemia is neuroprotective, indicating a crucial role for Ca²⁺-permeable AMPARs in ischemic cell death (16).In addition to delayed changes in AMPAR subunit composition as a result of altered mRNA expression, it was recently reported that Ca²⁺-permable, GluR2-lacking AMPARs are targeted to synaptic sites via membrane trafficking at much earlier times during OGD (17). This subunit rearrangement involves endocytosis of AMPARs containing GluR2 complexed with GluR1/3, followed by exocytosis of GluR2-lacking receptors containing GluR1/3 (17). However, the molecular mechanisms behind this trafficking event are unknown, and furthermore, it is not known whether these trafficking-mediated changes in AMPAR subunit composition contribute to delayed cell death.AMPAR trafficking is a well studied phenomenon because of its crucial involvement in long term depression (LTD) and long term potentiation (LTP), activity-dependent forms of synaptic plasticity thought to underlie learning and memory. AMPAR endocytosis, exocytosis, and more recently subunit-switching events (brought about by trafficking that involves endo/exocytosis) are central to the necessary changes in synaptic receptor complement (7, 18–20). It is possible that similar mechanisms regulate AMPAR trafficking during OGD.PICK1 is a PDZ and BAR (Bin-amphiphysin-Rus) domain-containing protein that binds, via the PDZ domain, to a number of membrane proteins including AMPAR subunits GluR2/3. This interaction is required for AMPAR internalization from the synaptic plasma membrane in response to Ca²⁺ influx via NMDAR activation in hippocampal neurons (21–23). This process is the major mechanism that underlies the reduction in synaptic strength in LTD. Furthermore, PICK1-mediated trafficking has recently emerged as a mechanism that regulates the GluR2 content of synaptic receptors, which in turn determines their Ca²⁺ permeability (7, 20). This is likely to be of profound importance in both plasticity and pathological mechanisms. Importantly, PICK1 overexpression has been shown to induce a shift in synaptic AMPAR subunit composition in hippocampal CA1 neurons, resulting in inwardly rectifying AMPAR EPSCs via reduced surface GluR2 and no change in GluR1 (24). This suggests that PICK1 may mediate the rapid switch in subunit composition occurring during OGD (17). Here, we demonstrate that the OGD-induced switch in AMPAR subunit composition is dependent on PICK1 PDZ interactions, and importantly, that this early trafficking event that occurs during OGD contributes to the signaling that results in delayed neuronal death.     

The role of the GluR2 subunit in AMPA receptor function and synaptic plasticity

The AMPA receptor (AMPAR) GluR2 subunit dictates the critical biophysical properties of the receptor, strongly influences receptor assembly and trafficking, and plays pivotal roles in a number of forms of long-term synaptic plasticity. Most neuronal AMPARs contain this critical subunit; however, in certain restricted neuronal populations and under certain physiological or pathological conditions, AMPARs that lack this subunit are expressed. There is a current surge of interest in such GluR2-lacking Ca2+-permeable AMPARs in how they affect the regulation of synaptic transmission. Here, we bring together recent data highlighting the novel and important roles of GluR2 in synaptic function and plasticity.     

Synaptic AMPA receptor exchange maintains bidirectional plasticity

Activity-dependent synaptic delivery of GluR1-, GluR2L-, and GluR4-containing AMPA receptors (-Rs) and removal of GluR2-containing AMPA-Rs mediate synaptic potentiation and depression, respectively. The obvious puzzle is how synapses maintain the capacity for bidirectional plasticity if different AMPA-Rs are utilized for potentiation and depression. Here, we show that synaptic AMPA-R exchange is essential for maintaining the capacity for bidirectional plasticity. The exchange process consists of activity-independent synaptic removal of GluR1-, GluR2L-, or GluR4-containing AMPA-Rs and refilling with GluR2-containing AMPA-Rs at hippocampal and cortical synapses in vitro and in intact brains. In GluR1 and GluR2 knockout mice, initiation or completion of synaptic AMPA-R exchange is compromised, respectively. The complementary AMPA-R removal and refilling events in the exchange process ultimately maintain synaptic strength unchanged, but their long rate time constants ( approximately 15-18 hr) render transmission temporarily depressed in the middle of the exchange. These results suggest that the previously hypothesized "slot" proteins, rather than AMPA-Rs, code and maintain transmission efficacy at central synapses.     

Glutamate receptors on dopamine neurons control the persistence of cocaine seeking

Cocaine strengthens excitatory synapses onto midbrain dopamine neurons through the synaptic delivery of GluR1-containing AMPA receptors. This cocaine-evoked plasticity depends on NMDA receptor activation, but its behavioral significance in the context of addiction remains elusive. Here, we generated mice lacking the GluR1, GluR2, or NR1 receptor subunits selectively in dopamine neurons. We report that in midbrain slices of cocaine-treated mice, synaptic transmission was no longer strengthened when GluR1 or NR1 was abolished, while in the respective mice the drug still induced normal conditioned place preference and locomotor sensitization. In contrast, extinction of drug-seeking behavior was absent in mice lacking GluR1, while in the NR1 mutant mice reinstatement was abolished. In conclusion, cocaine-evoked synaptic plasticity does not mediate concurrent short-term behavioral effects of the drug but may initiate adaptive changes eventually leading to the persistence of drug-seeking behavior.     

Localized Ca2+ entry preferentially effects protein dephosphorylation, phosphorylation, and glutamate release.

The release of neurotransmitter glutamate from isolated nerve terminals (synaptosomes) was found to be tightly coupled to the entry of Ca2+ through voltage-dependent Ca2+ channels, but is relatively unresponsive to "bulk" increases in cytosolic Ca2+ concentrations ([Ca2+]c) effected by Ca2+ ionophore. Under the same conditions, this dependence on Ca2+ influx, specifically through Ca2+ channels, was also seen for the dephosphorylation of a 96-kDa protein, (P96), present in the nerve terminals, as well as the phosphorylation of proteins migrating at 75 kDa (P75), corresponding to the synapsins, a group of well characterized synaptic vesicle-associated proteins. P96 dephosphorylation, following Ca2+ influx, was persistent and insensitive to the phosphatase inhibitor okadaic acid, suggesting a phosphatase other than protein phosphatase 1 and 2A as being responsible. Perhaps through the same phosphatase activity the increase in P75 phosphorylation was rapidly reversed with a time course similar to P96 dephosphorylation. When release, P96 dephosphorylation, and P75 phosphorylation were considered as functions of the [Ca2+]c increases achieved by depolarization and Ca2+ ionophore, there was no correlation of any of these with the overall concentration of Ca2+ in the cytosol. Since the fura-2 method used to measure [Ca2+] gives an averaged [Ca2+]c, these results imply that the release and protein dephosphorylation events are functionally coupled to local [Ca2+]c, in the immediate vicinity of Ca2+ channels. The reported clustering of the latter at the active zone area of the synapse and the parallelism between synaptic vesicle exocytosis and the phosphorylation of synaptic vesicle-associated proteins (p75:synapsins Ia/Ib), suggests that P96 may be similarly localized at the active zone area and, therefore, may be of significance in a modulatory role in glutamate release.     

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.