Clustering of CaV 1.3 L-type calcium channels by Shank3.

Clustering of L-type voltage-gated Ca2+ channels (LTCCs) in the plasma membrane is increasingly implicated in creating highly localized Ca2+ signaling nanodomains. For example, neuronal LTCC activation can increase phosphorylation of the nuclear CREB transcription factor by increasing Ca2+ concentrations within a nanodomain close to the channel, without requiring bulk Ca2+ increases in the cytosol or nucleus. However, the molecular basis for LTCC clustering is poorly understood. The postsynaptic scaffolding protein Shank3 specifically associates with one of the major neuronal LTCCs, the CaV 1.3 calcium channel, and is required for optimal LTCC-dependent excitation-transcription coupling. Here, we co-expressed CaV 1.3 α1 subunits with two distinct epitope-tags with or without Shank3 in HEK cells. Co-immunoprecipitation studies using the cell lysates revealed that Shank3 can assemble complexes containing multiple CaV 1.3 α1 subunits under basal conditions. Moreover, CaV 1.3 LTCC complex formation was facilitated by CaV ß subunits (ß3 and ß2a), which also interact with Shank3. Shank3 interactions with CaV 1.3 LTCCs and multimeric CaV 1.3 LTCC complex assembly were disrupted following the addition of Ca2+ to cell lysates, perhaps simulating conditions within an activated CaV 1.3 LTCC nanodomain. In intact HEK293T cells, co-expression of Shank3 enhanced the intensity of membrane-localized CaV 1.3 LTCC clusters under basal conditions, but not after Ca2+ channel activation. Live cell imaging studies also revealed that Ca2+ influx through LTCCs disassociated Shank3 from CaV 1.3 LTCCs clusters and reduced the CaV 1.3 cluster intensity. Deletion of the Shank3 PDZ domain prevented both binding to CaV 1.3 and the changes in multimeric CaV 1.3 LTCC complex assembly in vitro and in HEK293 cells. Finally, we found that shRNA knock-down of Shank3 expression in cultured rat primary hippocampal neurons reduced the intensity of surface-localized CaV 1.3 LTCC clusters in dendrites. Taken together, our findings reveal a novel molecular mechanism contributing to neuronal LTCC clustering under basal conditions.

Proteolytic regulation of calcium channels - avoiding controversy.

The publication of papers containing data obtained with suboptimal rigor in the experimental design and choice of key reagents, such as antibodies, can result in a lack of reproducibility and generate controversy that can both needlessly divert resources and, in some cases, damage public perception of the scientific enterprise. This exemplary paper by Buonarati et al. (2018)1 shows how a previously published, potentially important paper on calcium channel regulation falls short of the necessary mark, and aims to resolve the resulting controversy.

Neuronal L-Type Calcium Channel Signaling to the Nucleus Requires a Novel CaMKIIα-Shank3 Interaction.

The activation of neuronal plasma membrane Ca2+ channels stimulates many intracellular responses. Scaffolding proteins can preferentially couple specific Ca2+ channels to distinct downstream outputs, such as increased gene expression, but the molecular mechanisms that underlie the exquisite specificity of these signaling pathways are incompletely understood. Here, we show that complexes containing CaMKII and Shank3, a postsynaptic scaffolding protein known to interact with L-type calcium channels (LTCCs), can be specifically coimmunoprecipitated from mouse forebrain extracts. Activated purified CaMKIIα also directly binds Shank3 between residues 829 and 1130. Mutation of Shank3 residues 949Arg-Arg-Lys951 to three alanines disrupts CaMKII binding in vitro and CaMKII association with Shank3 in heterologous cells. Our shRNA/rescue studies revealed that Shank3 binding to both CaMKII and LTCCs is important for increased phosphorylation of the nuclear CREB transcription factor and expression of c-Fos induced by depolarization of cultured hippocampal neurons. Thus, this novel CaMKII-Shank3 interaction is essential for the initiation of a specific long-range signal from LTCCs in the plasma membrane to the nucleus that is required for activity-dependent changes in neuronal gene expression during learning and memory.SIGNIFICANCE STATEMENT Precise neuronal expression of genes is essential for normal brain function. Proteins involved in signaling pathways that underlie activity-dependent gene expression, such as CaMKII, Shank3, and L-type calcium channels, are often mutated in multiple neuropsychiatric disorders. Shank3 and CaMKII were previously shown to bind L-type calcium channels, and we show here that Shank3 also binds to CaMKII. Our data show that each of these interactions is required for depolarization-induced phosphorylation of the CREB nuclear transcription factor, which stimulates the expression of c-Fos, a neuronal immediate early gene with key roles in synaptic plasticity, brain development, and behavior.

Cyclic AMP-dependent protein kinase and D1 dopamine receptors regulate diacylglycerol lipase-α and synaptic 2-arachidonoyl glycerol signaling.

Brain endocannabinoids serve as retrograde neurotransmitters, being synthesized in post-synaptic neurons "on demand" and released to bind pre-synaptic cannabinoid receptors and suppress glutamatergic or GABAergic transmission. The most abundant brain endocannabinoid, 2 arachidonoyl glycerol (2-AG), is primarily synthesized by diacylglycerol lipase-α (DGLα), which is activated by poorly understood mechanisms in response to calcium influx following post-synaptic depolarization and/or the activation of Gq -coupled group 1 metabotropic glutamate receptors. However, the impact of other neurotransmitters and their downstream signaling pathways on synaptic 2-AG signaling has not been intensively studied. Here, we found that DGLα activity in membrane fractions from transfected HEK293T cells was significantly increased by in vitro phosphorylation using cyclic AMP-dependent protein kinase (PKA). Moreover, PKA directly phosphorylated DGLα at Ser798 in vitro. Elevation of cAMP levels in HEK293 cells expressing DGLα increased Ser798 phosphorylation, as detected using a phospho-Ser798-specific antibody, and enhanced DGLα activity; this in situ enhancement of DGLα activity was prevented by mutation of Ser798 to Ala. We investigated the impact of PKA on synaptic 2-AG mobilization in mouse striatal slices by manipulating D1-dopamine receptor (D1R) signaling and assessing depolarization-induced suppression of excitation, a DGLα- and 2-AG-dependent form of short-term synaptic depression. The magnitude of depolarization-enhanced suppression of excitation in direct pathway medium spiny neurons was increased by pre-incubation with a D1R agonist, and this enhancement was blocked by post-synaptic inhibition of PKA. Taken together, these findings provide new molecular insights into the complex mechanisms regulating synaptic endocannabinoid signaling.

CaMKIIα phosphorylation of Shank3 modulates ABI1-Shank3 interaction.

Protein-protein interactions can be modulated by phosphorylation of either binding partner, thereby altering subcellular localization and/or physiological function. Shank3, a master postsynaptic scaffolding protein that controls the developmental maturation of excitatory synapses, was recently shown to be phosphorylated by Protein Kinase A (PKA) at Ser685 in vivo. Mutation of Shank3 Ser685 was shown to modulate the binding of Abelson interactor 1 (ABI1), a component of the WAVE regulatory complex for actin remodeling, but a direct effect of Ser685 phosphorylation on ABI1 binding was not investigated. Here, we demonstrate that Ca2+/calmodulin-dependent protein kinase II alpha (CaMKIIα) also phosphorylates Shank3 at Ser685. Mutation of Ser685 to phospho-null alanine (S685A) prevented both CaMKIIα and PKA phosphorylation of a GST-Shank3 fusion protein. The co-immunoprecipitation of ABI1 with Shank3 from HEK293 cell extracts is reduced by mutation of Ser685 to either Ala or Asp. However, pre-phosphorylation of GST-Shank3 by purified CaMKIIα significantly increased binding of ABI1, and this effect was abrogated by Ser685 to Ala mutation in GST-Shank3. Taken together, our data suggest that neuronal ABI1-Shank3 interactions may be convergently regulated by Shank3 Ser685 phosphorylation in response to both Ca2+ and cAMP signaling, potentially modulating dendritic spine morphology.

Activated CaMKIIα Binds to the mGlu5 Metabotropic Glutamate Receptor and Modulates Calcium Mobilization.

Ca2+/calmodulin-dependent protein kinase II (CaMKII) and metabotropic glutamate receptor 5 (mGlu5) are critical signaling molecules in synaptic plasticity and learning/memory. Here, we demonstrate that mGlu5 is present in CaMKIIα complexes isolated from mouse forebrain. Further in vitro characterization showed that the membrane-proximal region of the C-terminal domain (CTD) of mGlu5a directly interacts with purified Thr286-autophosphorylated (activated) CaMKIIα However, the binding of CaMKIIα to this CTD fragment is reduced by the addition of excess Ca2+/calmodulin or by additional CaMKIIα autophosphorylation at non-Thr286 sites. Furthermore, in vitro binding of CaMKIIα is dependent on a tribasic residue motif Lys-Arg-Arg (KRR) at residues 866-868 of the mGlu5a-CTD, and mutation of this motif decreases the coimmunoprecipitation of CaMKIIα with full-length mGlu5a expressed in heterologous cells by about 50%. The KRR motif is required for two novel functional effects of coexpressing constitutively active CaMKIIα with mGlu5a in heterologous cells. First, cell-surface biotinylation studies showed that CaMKIIα increases the surface expression of mGlu5a Second, using Ca2+ fluorimetry and single-cell Ca2+ imaging, we found that CaMKIIα reduces the initial peak of mGlu5a-mediated Ca2+ mobilization by about 25% while doubling the relative duration of the Ca2+ signal. These findings provide new insights into the physical and functional coupling of these key regulators of postsynaptic signaling.

Introduction to the Thematic Minireview Series: Brain glycogen metabolism.

The synthesis of glycogen allows for efficient intracellular storage of glucose molecules in a soluble form that can be rapidly released to enter glycolysis in response to energy demand. Intensive studies of glucose and glycogen metabolism, predominantly in skeletal muscle and liver, have produced innumerable insights into the mechanisms of hormone action, resulting in the award of several Nobel Prizes over the last one hundred years. Glycogen is actually present in all cells and tissues, albeit at much lower levels than found in muscle or liver. However, metabolic and physiological roles of glycogen in other tissues are poorly understood. This series of Minireviews summarizes what is known about the enzymes involved in brain glycogen metabolism and studies that have linked glycogen metabolism to multiple brain functions involving metabolic communication between astrocytes and neurons. Recent studies unexpectedly linking some forms of epilepsy to mutations in two poorly understood proteins involved in glycogen metabolism are also reviewed.

Role of Striatal Direct Pathway 2-Arachidonoylglycerol Signaling in Sociability and Repetitive Behavior.

BACKGROUND: Endocannabinoid signaling plays an important role in regulating synaptic transmission in the striatum, a brain region implicated as a central node of dysfunction in autism spectrum disorder. Deficits in signaling mediated by the endocannabinoid 2-arachidonoylglycerol (2-AG) have been reported in mouse models of autism spectrum disorder, but a causal role for striatal 2-AG deficiency in phenotypes relevant to autism spectrum disorder has not been explored. METHODS: Using conditional knockout mice, we examined the electrophysiological, biochemical, and behavioral effects of 2-AG deficiency by deleting its primary synthetic enzyme, diacylglycerol lipase α (DGLα), from dopamine D1 receptor-expressing or adenosine A2a receptor-expressing medium spiny neurons (MSNs) to determine the role of 2-AG signaling in striatal direct or indirect pathways, respectively. We then used viral-mediated deletion of DGLα to study the effects of 2-AG deficiency in the ventral and dorsal striatum. RESULTS: Targeted deletion of DGLα from direct-pathway MSNs caused deficits in social interaction, excessive grooming, and decreased exploration of a novel environment. In contrast, deletion from indirect-pathway MSNs had no effect on any measure of behavior examined. Loss of 2-AG in direct-pathway MSNs also led to increased glutamatergic drive, which is consistent with a loss of retrograde feedback inhibition. Subregional DGLα deletion from the dorsal striatum produced deficits in social interaction, whereas deletion from the ventral striatum resulted in repetitive grooming. CONCLUSIONS: These data suggest a role for 2-AG deficiency in social deficits and repetitive behavior, and they demonstrate a key role for 2-AG in regulating striatal direct-pathway MSNs.

A novel mechanism for Ca2+/calmodulin-dependent protein kinase II targeting to L-type Ca2+ channels that initiates long-range signaling to the nucleus.

Neuronal excitation can induce new mRNA transcription, a phenomenon called excitation-transcription (E-T) coupling. Among several pathways implicated in E-T coupling, activation of voltage-gated L-type Ca2+ channels (LTCCs) in the plasma membrane can initiate a signaling pathway that ultimately increases nuclear CREB phosphorylation and, in most cases, expression of immediate early genes. Initiation of this long-range pathway has been shown to require recruitment of Ca2+-sensitive enzymes to a nanodomain in the immediate vicinity of the LTCC by an unknown mechanism. Here, we show that activated Ca2+/calmodulin-dependent protein kinase II (CaMKII) strongly interacts with a novel binding motif in the N-terminal domain of CaV1 LTCC α1 subunits that is not conserved in CaV2 or CaV3 voltage-gated Ca2+ channel subunits. Mutations in the CaV1.3 α1 subunit N-terminal domain or in the CaMKII catalytic domain that largely prevent the in vitro interaction also disrupt CaMKII association with intact LTCC complexes isolated by immunoprecipitation. Furthermore, these same mutations interfere with E-T coupling in cultured hippocampal neurons. Taken together, our findings define a novel molecular interaction with the neuronal LTCC that is required for the initiation of a long-range signal to the nucleus that is critical for learning and memory.

The Atypical MAP Kinase SWIP-13/ERK8 Regulates Dopamine Transporters through a Rho-Dependent Mechanism.

The neurotransmitter dopamine (DA) regulates multiple behaviors across phylogeny, with disrupted DA signaling in humans associated with addiction, attention-deficit/ hyperactivity disorder, schizophrenia, and Parkinson's disease. The DA transporter (DAT) imposes spatial and temporal limits on DA action, and provides for presynaptic DA recycling to replenish neurotransmitter pools. Molecular mechanisms that regulate DAT expression, trafficking, and function, particularly in vivo, remain poorly understood, though recent studies have implicated rho-linked pathways in psychostimulant action. To identify genes that dictate the ability of DAT to sustain normal levels of DA clearance, we pursued a forward genetic screen in Caenorhabditis elegans based on the phenotype swimming-induced paralysis (Swip), a paralytic behavior observed in hermaphrodite worms with loss-of-function dat-1 mutations. Here, we report the identity of swip-13, which encodes a highly conserved ortholog of the human atypical MAP kinase ERK8. We present evidence that SWIP-13 acts presynaptically to insure adequate levels of surface DAT expression and DA clearance. Moreover, we provide in vitro and in vivo evidence supporting a conserved pathway involving SWIP-13/ERK8 activation of Rho GTPases that dictates DAT surface expression and function.SIGNIFICANCE STATEMENT Signaling by the neurotransmitter dopamine (DA) is tightly regulated by the DA transporter (DAT), insuring efficient DA clearance after release. Molecular networks that regulate DAT are poorly understood, particularly in vivo Using a forward genetic screen in the nematode Caenorhabditis elegans, we implicate the atypical mitogen activated protein kinase, SWIP-13, in DAT regulation. Moreover, we provide in vitro and in vivo evidence that SWIP-13, as well as its human counterpart ERK8, regulate DAT surface availability via the activation of Rho proteins. Our findings implicate a novel pathway that regulates DA synaptic availability and that may contribute to risk for disorders linked to perturbed DA signaling. Targeting this pathway may be of value in the development of therapeutics in such disorders.

Densin-180 Controls the Trafficking and Signaling of L-Type Voltage-Gated Cav1.2 Ca2+ Channels at Excitatory Synapses.

Voltage-gated Cav1.2 and Cav1.3 (L-type) Ca2+ channels regulate neuronal excitability, synaptic plasticity, and learning and memory. Densin-180 (densin) is an excitatory synaptic protein that promotes Ca2+-dependent facilitation of voltage-gated Cav1.3 Ca2+ channels in transfected cells. Mice lacking densin (densin KO) exhibit defects in synaptic plasticity, spatial memory, and increased anxiety-related behaviors-phenotypes that more closely match those in mice lacking Cav1.2 than Cav1.3. Therefore, we investigated the functional impact of densin on Cav1.2. We report that densin is an essential regulator of Cav1.2 in neurons, but has distinct modulatory effects compared with its regulation of Cav1.3. Densin binds to the N-terminal domain of Cav1.2, but not that of Cav1.3, and increases Cav1.2 currents in transfected cells and in neurons. In transfected cells, densin accelerates the forward trafficking of Cav1.2 channels without affecting their endocytosis. Consistent with a role for densin in increasing the number of postsynaptic Cav1.2 channels, overexpression of densin increases the clustering of Cav1.2 in dendrites of hippocampal neurons in culture. Compared with wild-type mice, the cell surface levels of Cav1.2 in the brain, as well as Cav1.2 current density and signaling to the nucleus, are reduced in neurons from densin KO mice. We conclude that densin is an essential regulator of neuronal Cav1 channels and ensures efficient Cav1.2 Ca2+ signaling at excitatory synapses.SIGNIFICANCE STATEMENT The number and localization of voltage-gated Cav Ca2+ channels are crucial determinants of neuronal excitability and synaptic transmission. We report that the protein densin-180 is highly enriched at excitatory synapses in the brain and enhances the cell surface trafficking and postsynaptic localization of Cav1.2 L-type Ca2+ channels in neurons. This interaction promotes coupling of Cav1.2 channels to activity-dependent gene transcription. Our results reveal a mechanism that may contribute to the roles of Cav1.2 in regulating cognition and mood.

Endocannabinoid signalling modulates susceptibility to traumatic stress exposure.

Stress is a ubiquitous risk factor for the exacerbation and development of affective disorders including major depression and posttraumatic stress disorder. Understanding the neurobiological mechanisms conferring resilience to the adverse consequences of stress could have broad implications for the treatment and prevention of mood and anxiety disorders. We utilize laboratory mice and their innate inter-individual differences in stress-susceptibility to demonstrate a critical role for the endogenous cannabinoid 2-arachidonoylglycerol (2-AG) in stress-resilience. Specifically, systemic 2-AG augmentation is associated with a stress-resilient phenotype and enhances resilience in previously susceptible mice, while systemic 2-AG depletion or CB1 receptor blockade increases susceptibility in previously resilient mice. Moreover, stress-resilience is associated with increased phasic 2-AG-mediated synaptic suppression at ventral hippocampal-amygdala glutamatergic synapses and amygdala-specific 2-AG depletion impairs successful adaptation to repeated stress. These data indicate amygdala 2-AG signalling mechanisms promote resilience to adverse effects of acute traumatic stress and facilitate adaptation to repeated stress exposure.

Reduced bioavailable manganese causes striatal urea cycle pathology in Huntington's disease mouse model.

Huntington's disease (HD) is caused by a mutation in the huntingtin gene (HTT), resulting in profound striatal neurodegeneration through an unknown mechanism. Perturbations in the urea cycle have been reported in HD models and in HD patient blood and brain. In neurons, arginase is a central urea cycle enzyme, and the metal manganese (Mn) is an essential cofactor. Deficient biological responses to Mn, and reduced Mn accumulation have been observed in HD striatal mouse and cell models. Here we report in vivo and ex vivo evidence of a urea cycle metabolic phenotype in a prodromal HD mouse model. Further, either in vivo or in vitro Mn supplementation reverses the urea-cycle pathology by restoring arginase activity. We show that Arginase 2 (ARG2) is the arginase enzyme present in these mouse brain models, with ARG2 protein levels directly increased by Mn exposure. ARG2 protein is not reduced in the prodromal stage, though enzyme activity is reduced, indicating that altered Mn bioavailability as a cofactor leads to the deficient enzymatic activity. These data support a hypothesis that mutant HTT leads to a selective deficiency of neuronal Mn at an early disease stage, contributing to HD striatal urea-cycle pathophysiology through an effect on arginase activity.

A Novel Human CAMK2A Mutation Disrupts Dendritic Morphology and Synaptic Transmission, and Causes ASD-Related Behaviors.

Characterizing the functional impact of novel mutations linked to autism spectrum disorder (ASD) provides a deeper mechanistic understanding of the underlying pathophysiological mechanisms. Here we show that a de novo Glu183 to Val (E183V) mutation in the CaMKIIα catalytic domain, identified in a proband diagnosed with ASD, decreases both CaMKIIα substrate phosphorylation and regulatory autophosphorylation, and that the mutated kinase acts in a dominant-negative manner to reduce CaMKIIα-WT autophosphorylation. The E183V mutation also reduces CaMKIIα binding to established ASD-linked proteins, such as Shank3 and subunits of l-type calcium channels and NMDA receptors, and increases CaMKIIα turnover in intact cells. In cultured neurons, the E183V mutation reduces CaMKIIα targeting to dendritic spines. Moreover, neuronal expression of CaMKIIα-E183V increases dendritic arborization and decreases both dendritic spine density and excitatory synaptic transmission. Mice with a knock-in CaMKIIα-E183V mutation have lower total forebrain CaMKIIα levels, with reduced targeting to synaptic subcellular fractions. The CaMKIIα-E183V mice also display aberrant behavioral phenotypes, including hyperactivity, social interaction deficits, and increased repetitive behaviors. Together, these data suggest that CaMKIIα plays a previously unappreciated role in ASD-related synaptic and behavioral phenotypes.SIGNIFICANCE STATEMENT Many autism spectrum disorder (ASD)-linked mutations disrupt the function of synaptic proteins, but no single gene accounts for >1% of total ASD cases. The molecular networks and mechanisms that couple the primary deficits caused by these individual mutations to core behavioral symptoms of ASD remain poorly understood. Here, we provide the first characterization of a mutation in the gene encoding CaMKIIα linked to a specific neuropsychiatric disorder. Our findings demonstrate that this ASD-linked de novo CAMK2A mutation disrupts multiple CaMKII functions, induces synaptic deficits, and causes ASD-related behavioral alterations, providing novel insights into the synaptic mechanisms contributing to ASD.

Chronic intermittent alcohol disrupts the GluN2B-associated proteome and specifically regulates group I mGlu receptor-dependent long-term depression.

N-Methyl-d-aspartate receptors (NMDARs) are major targets of both acute and chronic alcohol, as well as regulators of plasticity in a number of brain regions. Aberrant plasticity may contribute to the treatment resistance and high relapse rates observed in alcoholics. Recent work suggests that chronic alcohol treatment preferentially modulates both the expression and subcellular localization of NMDARs containing the GluN2B subunit. Signaling through synaptic and extrasynaptic GluN2B-NMDARs has already been implicated in the pathophysiology of various other neurological disorders. NMDARs interact with a large number of proteins at the glutamate synapse, and a better understanding of how alcohol modulates this proteome is needed. We employed a discovery-based proteomic approach in subcellular fractions of hippocampal tissue from chronic intermittent alcohol (CIE)-exposed C57Bl/6J mice to gain insight into alcohol-induced changes in GluN2B signaling complexes. Protein enrichment analyses revealed changes in the association of post-synaptic proteins, including scaffolding, glutamate receptor and PDZ-domain binding proteins with GluN2B. In particular, GluN2B interaction with metabotropic glutamate (mGlu)1/5 receptor-dependent long-term depression (LTD)-associated proteins such as Arc and Homer 1 was increased, while GluA2 was decreased. Accordingly, we found a lack of mGlu1/5 -induced LTD while α1 -adrenergic receptor-induced LTD remained intact in hippocampal CA1 following CIE. These data suggest that CIE specifically disrupts mGlu1/5 -LTD, representing a possible connection between NMDAR and mGlu receptor signaling. These studies not only demonstrate a new way in which alcohol can modulate plasticity in the hippocampus but also emphasize the utility of this discovery-based proteomic approach to generate new hypotheses regarding alcohol-related mechanisms.

CaMKII-mediated phosphorylation of GluN2B regulates recombinant NMDA receptor currents in a chloride-dependent manner.

Some forms of long-term synaptic plasticity require docking of Ca2+/calmodulin-dependent protein kinase II α (CaMKIIα) to residues 1290-1309 within the intracellular C-terminal tail of the N-methyl-d-aspartate (NMDA) receptor GluN2B subunit. The phosphorylation of Ser1303 within this region destabilizes CaMKII binding. Interestingly, Ser1303 is a substrate for CaMKII itself, as well as PKC and DAPK1, but these kinases have been reported to have contradictory effects on the activity of GluN2B-containing NMDA receptors. Here, we re-assessed the effect of CaMKII on NMDA receptor desensitization in heterologous cells, as measured by the ratio of steady-state to peak currents induced during 3s agonist applications. CaMKIIα co-expression or infusion of constitutively active CaMKII limits the extent of desensitization and preserves current amplitude with repeated stimulation of recombinant GluN1A/GluN2B when examined using low intracellular chloride (Cl-) levels, characteristic of neurons beyond the first postnatal week. In contrast, CaMKIIα enhances the acute rate and extent of desensitization when intracellular Cl- concentrations are high. The apparent dependence of CaMKIIα effects on NMDA receptor desensitization on Cl- concentrations is consistent with the presence of a Ca2+-activated Cl- conductance endogenous to HEK 293 cells, which was confirmed by photolysis of caged-Ca2+. However, Ca2+-activated Cl- conductances are unaffected by CaMKIIα expression, indicating that CaMKII affects agonist-induced whole cell currents via modulation of the NMDA receptor. In support of this idea, CaMKIIα modulation of GluN2B-NMDA receptors is abrogated by the phospho-null mutation of Ser1303 in GluN2B to alanine and occluded by phospho-mimetic mutation of Ser1303 to aspartate regardless of intracellular Cl- concentration. Thus, CaMKII-mediated phosphorylation of GluN2B-containing NMDA receptors reduces desensitization at physiological (low) intracellular Cl-, perhaps serving as a feed-forward mechanism to sustain NMDA-mediated Ca2+ entry and continued CaMKII activation during learning and memory.

Changes in the Adult GluN2B Associated Proteome following Adolescent Intermittent Ethanol Exposure.

Adolescent alcohol use is the strongest predictor for alcohol use disorders. In rodents, adolescents have distinct responses to acute ethanol, and prolonged alcohol exposure during adolescence can maintain these phenotypes into adulthood. One brain region that is particularly sensitive to the effects of both acute and chronic ethanol exposure is the hippocampus. Adolescent intermittent ethanol exposure (AIE) produces long lasting changes in hippocampal synaptic plasticity and dendritic morphology, as well as in the susceptibility to acute ethanol-induced spatial memory impairment. Given the pattern of changes in hippocampal structure and function, one potential target for these effects is the ethanol sensitive GluN2B subunit of the NMDA receptor, which is known to be involved in synaptic plasticity and dendritic morphology. Thus we sought to determine if there were persistent changes in hippocampal GluN2B signaling cascades following AIE. We employed a previously validated GluN2B-targeted proteomic strategy that was used to identify novel signaling mechanisms altered by chronic ethanol exposure in the adult hippocampus. We collected adult hippocampal tissue (P70) from rats that had been given 2 weeks of AIE from P30-45. Tissue extracts were fractionated into synaptic and non-synaptic pools, immuno-precipitated for GluN2B, and then analyzed using proteomic methods. We detected a large number of proteins associated with GluN2B. AIE produced significant changes in the association of many proteins with GluN2B in both synaptic and non-synaptic fractions. Intriguingly the number of proteins changed in the non-synaptic fraction was double that found in the synaptic fraction. Some of these proteins include those involved in glutamate signaling cytoskeleton rearrangement, calcium signaling, and plasticity. Disruptions in these pathways may contribute to the persistent cellular and behavioral changes found in the adult hippocampus following AIE. Further, the robust change in non-synaptic proteins suggests that AIE may prime this signaling pathway for future ethanol exposures in adulthood.