Calcium- and calmodulin-dependent protein kinase II (CaMKII) and glutamate receptors are integrally involved in forms of synaptic plasticity that may underlie learning and memory. In the simplest model for long-term potentiation, CaMKII is activated by Ca2+ influx through NMDA (N-methyl-D-aspartate) receptors and then potentiates synaptic efficacy by inducing synaptic insertion and increased single-channel conductance of AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors. Here we show that regulated CaMKII interaction with two sites on the NMDA receptor subunit NR2B provides a mechanism for the glutamate-induced translocation of the kinase to the synapse in hippocampal neurons. This interaction can lead to additional forms of potentiation by: facilitated CaMKII response to synaptic Ca2+; suppression of inhibitory autophosphorylation of CaMKII; and, most notably, direct generation of sustained Ca2+/calmodulin (CaM)-independent (autonomous) kinase activity by a mechanism that is independent of the phosphorylation state. Furthermore, the interaction leads to trapping of CaM that may reduce down-regulation of NMDA receptor activity. CaMKII-NR2B interaction may be prototypical for direct activation of a kinase by its targeting protein.

Acid-sensing ion channel (ASIC

Rapid glutamatergic synaptic transmission is mediated by ionotropic glutamate receptors and depends on their precise localization at postsynaptic membranes opposing the presynaptic neurotransmitter release sites. Postsynaptic localization of N-methyl-D-aspartatetype glutamate receptors may be mediated by the synapse-associated proteins (SAPs) SAP90, SAP102, and chapsyn-110. SAPs contain three PDZ domains that can interact with the C termini of proteins such as N-methyl-D-aspartate receptor subunits that carry a serine or threonine at the -2 position and a valine, isoleucine, or leucine at the very C terminus (position 0). We now show that SAP97, a SAP whose function at the synapse has been unclear, is associated with ␣-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-type glutamate receptors. AMPA receptors are probably tetramers and are formed by two or more of the four AMPA receptor subunits GluR1-4. GluR1 possesses a C-terminal consensus sequence for interactions with PDZ domains of SAPs. SAP97 was present in AMPA receptor complexes immunoprecipitated from detergent extracts of rat brain. After treatment of rat brain membrane fractions with the cross-linker dithiobis(succinimidylpropionate) and solubilization with sodium dodecylsulfate, SAP97 was associated with GluR1 but not GluR2 or GluR3. In vitro experiments with recombinant proteins indicate that SAP97 specifically associates with the C terminus of GluR1 but not other AMPA receptor subunits. Our findings suggest that SAP97 may be involved in localizing AMPA receptors at postsynaptic sites through its interaction with the GluR1 subunit.The prevailing excitatory neurotransmitter in the mammalian brain is glutamate (1, 2). Upon its release from presynaptic sites, this neurotransmitter binds to ionotropic glutamate receptors that mediate rapid excitatory synaptic transmission in the mammalian brain (1, 2). Several immuno-electron microscopic studies have demonstrated that ionotropic glutamate receptors are clustered at postsynaptic sites of excitatory synapses (3-5). Two major glutamate receptor families exist, namely N-methyl-D-aspartate (NMDA) 1 receptors, which mediate Ca 2ϩ influx, and non-NMDA receptors, which are usually not Ca 2ϩ -permeable (1, 2, 6). Non-NMDA receptors are further divided into ␣-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors and kainate receptors. At low frequency, synaptic transmission normally depends nearly exclusively upon AMPA receptors. On the other hand, kainate and NMDA receptors require higher frequencies for activation. NMDA receptor-mediated Ca 2ϩ influx is necessary for different forms of synaptic plasticity, such as long term potentiation (1,7,8). At different synapses in the hippocampus and in other brain areas, a few bursts of high frequency electric stimulation that activate NMDA receptors induce a long lasting increase in synaptic transmission, the hallmark of long term potentiation.Glutamate receptors are thought to be heterotetramers consisting of homologous subunits (39). AMPA recepto...

The molecular basis of long-term potentiation (LTP), a long-lasting change in synaptic transmission, is of fundamental interest because of its implication in learning. Usually LTP depends on Ca 2؉ inf lux through postsynaptic N-methyl-D-aspartate (NMDA)-type glutamate receptors and subsequent activation of Ca 2؉ ͞calmodulin-dependent protein kinase II (CaMKII). For a molecular understanding of LTP it is crucial to know how CaMKII is localized to its postsynaptic targets because protein kinases often are targeted to their substrates by adapter proteins. Here we show that CaMKII directly binds to the NMDA receptor subunits NR1 and NR2B. Moreover, activation of CaMKII␣ by stimulation of NMDA receptors in forebrain slices increase this association. This interaction places CaMKII not only proximal to a major source of Ca 2؉ inf lux but also close to ␣-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-type glutamate receptors, which become phosphorylated upon stimulation of NMDA receptors in these forebrain slices. Identification of the postsynaptic adapter for CaMKII fills a critical gap in the understanding of LTP because CaMKII-mediated phosphorylation of AMPA receptors is an important step during LTP. Caϩ ͞calmodulin-dependent protein kinase II (CaMKII) mediates a variety of different cellular responses to Ca 2ϩ influx (1, 2). An important source of Ca 2ϩ influx into neurons is the N-methyl-D-aspartate (NMDA)-type glutamate receptor, which is activated by the excitatory neurotransmitter glutamate (2). NMDA-and ␣-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-type glutamate receptors are clustered at postsynaptic sites opposing presynaptic neurotransmitter release sites (3, 4). Brief trains of presynaptic highfrequency stimulation efficiently activate NMDA receptors (5), resulting in postsynaptic Ca 2ϩ influx and long-term potentiation (LTP). LTP is a long-lasting increase in neurotransmission thought to represent the physiological correlate of learning and memory (5, 6). The induction of NMDA receptor-dependent LTP requires activation of CaMKII in the postsynaptic neuron (6, 7). CaMKII is enriched at postsynaptic densities (8, 9), where it is well placed for activation by Ca 2ϩ influx through NMDA receptors and subsequent phosphorylation of neighboring AMPA receptors, an event contributing to LTP (7, 10). A kinase anchoring proteins, usually called AKAPs, place cyclic AMP-dependent protein kinase (PKA) next to selected substrates such as AMPA receptors (11), and receptors for activated C kinase (RACKs) are important for subcellular localization of different protein kinase C (PKC) isozymes (12). Crucial information about the subcellular targeting of CaMKII is lacking. NMDA receptors would be ideal postsynaptic adapter sites for CaMKII, where it would have full access to Ca 2ϩ influx through these receptors. Cortical NMDA receptors consist of one or two NR1 and two or three NR2A and 2B subunits whose C termini are intracellular (13-16). We show that CaMKII is directly associated with NR1 a...

Ca2؉ influx through N-methyl-D-aspartate (NMDA)-type glutamate receptors plays a pivotal role in synaptic plasticity during brain development as well as in mature brain. Cyclic AMP-dependent protein kinase (PKA) and members of the protein kinase C (PKC) family are also essential for various forms of synaptic plasticity and regulate the activity of different ion channels including NMDA and non-NMDA receptors. We now demonstrate that PKA and various PKC isoforms phosphorylate the NMDA receptor in vitro. Ionotropic glutamate receptors mediate fast synaptic transmission by glutamate, the prevailing excitatory neurotransmitter in the mammalian brain (1, 2). These glutamate receptors can be divided into two major families, N-methyl-D-aspartate (NMDA) 1 receptors and non-NMDA receptors, based on their pharmacological and electrophysiological properties as well as sequence identity (1-3). NMDA receptors are key participants in brain development (4, 5) and synaptic plasticity, which may underlie learning and memory (6 -8).Analogous to other ligand-gated ion channels of the nicotinic acetylcholine receptor superfamily, glutamate receptors are thought to consist of five different subunits that are homologous to each other (3, 9, 10). NMDA receptors are formed by NR1 subunits in various combinations with NR2A-NR2D subunits (11-13). The apparent mass, as determined by immunoblotting, is 110 -120 kDa for NR1 and 160 -190 kDa for NR2A-D (14 -19). Deglycosylation in vitro reduces the apparent M r of glutamate receptor subunits, usually by 10 -20 kDa. Immunoprecipitation experiments suggest that native NMDA receptors are pentamers made of one or two NR1 and two or three NR2A-D subunits (15,19,20). Functional NMDA receptors are formed when NR1 is expressed alone or in combination with NR2 subunits, but not when NR2 subunits are expressed without NR1 (11,12). Targeted disruption of the NR1 gene results in a lethal effect (21), while the disruption of the NR2A gene has been implicated in reduced hippocampal long term potentiation (LTP) in mice (22). As expected from their role in synaptic transmission, biochemical (23) and immunohistochemical studies demonstrate that glutamate receptors are specifically localized at postsynaptic sites of excitatory, but not inhibitory, synapses (17,18,24,25).Hydrophobicity plots identified four hydrophobic regions in NMDA and non-NMDA receptors termed M1-M4. By comparison with nicotinic acetylcholine receptors, these regions were originally thought to form four transmembrane segments so that the NH 3 ϩ -and COO Ϫ -terminal ends would both be located extracellularly. Recent studies on non-NMDA receptor subunits, however, demonstrated that only the NH 3 ϩ -terminal end is extracellular, followed by the transmembrane region M1; the M2 region, which loops only partially into the plasma membrane and back into the cytosol; the transmembrane regions M3 and M4; and finally the cytosolic COO Ϫ terminus (26 -28). NMDA receptor subunits probably adopt a similar membrane topology (16).NMDA receptor-mediated cu...

The ability to detect salt is critical for the survival of terrestrial animals. Based on amiloride-dependent inhibition, the receptors that detect salt have been postulated to be DEG/ENaC channels. We found the Drosophila DEG/ENaC genes Pickpocket11 (ppk11) and Pickpocket19 (ppk19) expressed in the larval taste-sensing terminal organ and in adults on the taste bristles of the labelum, the legs, and the wing margins. When we disrupted PPK11 or PPK19 function, larvae lost their ability to discriminate low concentrations of Na(+) or K(+) from water, and the electrophysiologic responses to low salt concentrations were attenuated. In both larvae and adults, disrupting PPK11 or PPK19 affected the behavioral response to high salt concentrations. In contrast, the response of larvae to sucrose, pH 3, and several odors remained intact. These results indicate that the DEG/ENaC channels PPK11 and PPK19 play a key role in detecting Na(+) and K(+) salts.

Epilepsy, a disorder of recurrent seizures, is a common and frequently devastating neurological condition. Available therapy is only symptomatic and often ineffective. Understanding epileptogenesis, the process by which a normal brain becomes epileptic, may help identify molecular targets for drugs that could prevent epilepsy. A number of acquired and genetic causes of this disorder have been identified, and various in vivo and in vitro models of epileptogenesis have been established. Here, we review current insights into the molecular signaling mechanisms underlying epileptogenesis, focusing on limbic epileptogenesis. Study of different models reveals that activation of various receptors on the surface of neurons can promote epileptogenesis; these receptors include ionotropic and metabotropic glutamate receptors as well as the TrkB neurotrophin receptor. These receptors are all found in the membrane of a discrete signaling domain within a particular type of cortical neuron--the dendritic spine of principal neurons. Activation of any of these receptors results in an increase Ca2+ concentration within the spine. Various Ca2+-regulated enzymes found in spines have been implicated in epileptogenesis; these include the nonreceptor protein tyrosine kinases Src and Fyn and a serine-threonine kinase [Ca2+-calmodulin-dependent protein kinase II (CaMKII)] and phosphatase (calcineurin). Cross-talk between astrocytes and neurons promotes increased dendritic Ca2+ and synchronous firing of neurons, a hallmark of epileptiform activity. The hypothesis is proposed that limbic epilepsy is a maladaptive consequence of homeostatic responses to increases of Ca2+ concentration within dendritic spines induced by abnormal neuronal activity.

Ca2؉ influx through the N-methyl-D-aspartate (NMDA)-type glutamate receptor leads to activation and postsynaptic accumulation of Ca 2؉ /calmodulin-dependent protein kinase II (CaMKII) and ultimately to long term potentiation, which is thought to be the physiological correlate of learning and memory. The NMDA receptor also serves as a CaMKII docking site in dendritic spines with high affinity binding sites located on its NR1 and NR2B subunits. We demonstrate that high affinity binding of CaMKII to NR1 requires autophosphorylation of Thr 286 . This autophosphorylation reduces the off rate to a level (t1 ⁄2 ‫؍‬ ϳ23 min) that is similar to that observed for dissociation of the T286D mutant CaMKII (t1 ⁄2 ‫؍‬ ϳ30