Calmodulin-dependent Kinase II Research Articles

Nitric Oxide and Zinc-Mediated Protein Assemblies Involved in Mu Opioid Receptor Signaling

Opioids are among the most effective analgesics in controlling the perception of intense pain, although their continuous use decreases their potency due to the development of tolerance. The glutamate N-methyl-D-aspartate (NMDA) receptor system is currently considered to be the most relevant functional antagonist of morphine analgesia. In the postsynapse of different brain regions the C terminus of the mu-opioid receptor (MOR) associates with NR1 subunits of NMDARs, as well as with a series of signaling proteins, such as neural nitric oxide synthase (nNOS)/nitric oxide (NO), protein kinase C (PKC), calcium and calmodulin-dependent kinase II (CaMKII) and the mitogen-activated protein kinases (MAPKs). NO is implicated in redox signaling and PKC falls under the regulation of zinc metabolism, suggesting that these signaling elements might participate in the regulation of MOR activity by the NMDAR. In this review, we discuss the influence of redox signaling in the mechanisms whose plasticity triggers opioid tolerance. Thus, the MOR C terminus assembles a series of signaling proteins around the homodimeric histidine triad nucleotide-binding protein 1 (HINT1). The NMDAR NR1 subunit and the regulator of G protein signaling RGSZ2 bind HINT1 in a zinc-independent manner, with RGSZ2 associating with nNOS and regulating MOR-induced production of NO. This NO acts on the RGSZ2 zinc finger, providing the zinc ions that are required for PKC/Raf-1 cysteine-rich domains to simultaneously bind to the histidines present in the HINT1 homodimer. The MOR-induced activation of phospholipase β (PLCβ) regulates PKC, which increases the reactive oxygen species (ROS) by acting on NOX/NADPH, consolidating the long-term PKC activation required to regulate the Raf-1/MAPK cascade and enhancing NMDAR function. Thus, RGSZ2 serves as a Redox Zinc Switch that converts NO signals into Zinc signals, thereby modulating Redox Sensor Proteins like PKCγ and Raf-1. Accordingly, redox-dependent and independent processes weave together to situate the MOR under the negative control of the NMDAR.

Nonspecific, Reversible Inhibition of Voltage-Gated Calcium Channels by CaMKII Inhibitor CK59

Investigation of kinase-related processes often uses pharmacological inhibition to reveal pathways in which kinases are involved. However, one concern about using such kinase inhibitors is their potential lack of specificity. Here, we report that the calcium-calmodulin-dependent kinase II (CaMKII) inhibitor CK59 inhibited multiple voltage-gated calcium channels, including the L-type channel during depolarization in a dose-dependent manner. The use of another CaMKII inhibitor, cell-permeable autocamtide-2 related inhibitory peptide II (Ant-AIP-II), failed to similarly decrease calcium current or entry in hippocampal cultures, as shown by ratiometric calcium imaging and whole-cell patch clamp electrophysiology. Notably, inhibition due to CK59 was reversible; washout of the drug brought calcium levels back to control values upon depolarization. Furthermore, the IC50 for CK59 was approximately 50 μM, which is only fivefold larger than the reported IC50 values for CaMKII inhibition. Similar nonspecific actions of other CaMKII inhibitors KN93 and KN62 have previously been reported. In the case of all three kinase inhibitors, the IC50 for calcium current inhibition falls near that of CaMKII inhibition. Our findings demonstrate that CK59 attenuates activity of voltage-gated calcium channels, and thus provide more evidence for caution when relying on pharmacological inhibition to examine kinase-dependent phenomena.

Molecular determinants for cardiovascular TRPC6 channel regulation by Ca2+/calmodulin‐dependent kinase II

The molecular mechanism underlying Ca(2+)/calmodulin (CaM)-dependent kinase II (CaMKII)-mediated regulation of the mouse transient receptor potential channel TRPC6 was explored by chimera, deletion and site-directed mutagenesis approaches. Induction of currents (ICCh) in TRPC6-expressing HEK293 cells by a muscarinic agonist carbachol (CCh; 100 μm) was strongly attenuated by a CaMKII-specific peptide, autocamtide-2-related inhibitory peptide (AIP; 10 μm). TRPC6/C7 chimera experiments showed that the TRPC6 C-terminal sequence is indispensable for ICCh to be sensitive to AIP-induced CaMKII inhibition. Further, deletion of a distal region (Gln(855)-Glu(877)) of the C-terminal CaM/inositol-1,4,5-trisphosphate receptor binding domain (CIRB) of TRPC6 was sufficient to abolish ICCh. Systematic alanine scanning for potential CaMKII phosphorylation sites revealed that Thr(487) was solely responsible for the activation of the TRPC6 channel by receptor stimulation. The abrogating effect of the alanine mutation of Thr(487) (T487A) was reproduced with other non-polar amino acids, namely glutamine or asparagine, while being partially rescued by phosphomimetic mutations with glutamate or aspartate. The cellular expression and distribution of TRPC6 channels did not significantly change with these mutations. Electrophysiological and immunocytochemical data with the Myc-tagged TRPC6 channel indicated that Thr(487) is most likely located at the intracellular side of the cell membrane. Overexpression of T487A caused significant reduction of endogenous TRPC6-like current induced by Arg(8)-vasopressin in A7r5 aortic myocytes. Based on these results, we propose that the optimal spatial arrangement of a C-terminal domain (presumably the distal CIRB region) around a single CaMKII phosphorylation site Thr(487) may be essential for CaMKII-mediated regulation of TRPC6 channels. This mechanism may be of physiological significance in a native environment such as in vascular smooth muscle cells.

Lycopene Inhibits Cyclooxygenase‐2 And Inflammatory Mediator Expression In Microglia

Microglia is the main immune defense in the central nervous system. Activation of microglia leads to production excessive inflammatory molecules and deleterious consequences for neuronal death. Lycopene, one of the major carotenoids present in tomatoes, shown to exert antioxidant properties and inhibition of cancer cell proliferation. However, the effect of lycopene on neuroinflammatory responses in microglia still remains unknown. In this study, we investigated the signaling pathways involves in lycopene‐inhibited cyclooxygenase‐2 (COX‐2) and inflammatory mediator expression in microglia cells. Here, we demonstrate that lycopene inhibites lipopolysaccharide (LPS)‐induced COX‐2 and inflammatory mediator expression through heme oxygenase‐1 (HO‐1) activation. Our data also demonstrates that stimulation of lycopene increases the phosphorylation of serine–threonine liver kinase B1( LKB1), Calcium/calmodulin‐dependent kinase II( CaMKII) and AMP‐activated protein kinase (AMPK) in microglia. Interestingly, we also found that activation of phosphorylation‐AMPK by lycopene accumulates in the nucleus in microglia. Moreover, pre‐incubated cells with HO‐1 or AMPK pharmacological inhibitors effectively reversed the inhibitory effect of lycopene on LPS‐induced COX‐2 expression. Furthermore, transfection cells with HO‐1 or AMPK siRNA significantly attenuated the inhibitory effect of lycopene as well. These findings suggest that lycopeneinhibites LPS‐induced COX‐2 expression is mediated by HO‐1 activation through the AMPK pathways. Our results also indicate that lycopene is a promising nature product and may be as a useful pharmacological agent for the treatment of neuroinflammation‐associated disorders.

Ca2+/calmodulin-dependent kinase II contributes to inhibitor of nuclear factor-kappa B kinase complex activation inHelicobacter pyloriinfection

Helicobacter pylori, a class I carcinogen, induces a proinflammatory response by activating the transcription factor nuclear factor-kappa B (NF-κB) in gastric epithelial cells. This inflammatory condition could lead to chronic gastritis, which is epidemiologically and biologically linked to the development of gastric cancer. So far, there exists no clear knowledge on how H. pylori induces the NF-κB-mediated inflammatory response. In our study, we investigated the role of Ca(2+) /calmodulin-dependent kinase II (CAMKII), calmodulin, protein kinases C (PKCs) and the CARMA3-Bcl10-MALT1 (CBM) complex in conjunction with H. pylori-induced activation of NF-κB via the inhibitor of nuclear factor-kappa B kinase (IKK) complex. We use specific inhibitors and/or RNA interference to assess the contribution of these components. Our results show that CAMKII and calmodulin contribute to IKK complex activation and thus to the induction of NF-κB in response to H. pylori infection, but not in response to TNF-α. Thus, our findings are specific for H. pylori infected cells. Neither the PKCs α, δ, θ, nor the CBM complex itself is involved in the activation of NF-κB by H. pylori. The contribution of CAMKII and calmodulin, but not PKCs/CBM to the induction of an inflammatory response by H. pylori infection augment the understanding of the molecular mechanism involved and provide potential new disease markers for the diagnosis of gastric inflammatory diseases including gastric cancer.

Drosophila Bestrophin-1 Currents Are Regulated by Phosphorylation via a CaMKII Dependent Mechanism

Cell swelling induced by hypo-osmotic stress results in activation of volume-regulated anion channels (VRAC) that drive a compensatory regulatory volume decrease. We have previously shown that the Best1 gene in Drosophila encodes a VRAC that is also activated by increases in intracellular Ca2+. The role of Best1 as a VRAC has recently been independently confirmed by the Clapham lab in an unbiased RNAi screen. Although dBest1 is clearly a volume-regulated channel, its mechanisms of regulation remain unknown. Here we investigate Drosophila Best1 (dBest1) regulation using the Drosophila S2 cell model system. Because dBest1 activates slowly after establishing whole-cell recording, we tested the hypothesis that the channel is activated by phosphorylation. Two experiments indicate that phosphorylation is required for dBest1 activation: nonspecific protein kinase inhibitors or intracellular perfusion with the non-hydrolyzable ATP analog AMP-PNP dramatically reduce the amplitude of dBest1 currents. Furthermore, intracellular perfusion with ATP-γ-S augments channel activation. The kinase responsible for dBest1 activation is likely Ca2+/calmodulin dependent kinase II (CaMKII), because specific inhibitors of this kinase dramatically inhibit dBest1 current activation. Neither specific PKA inhibitors nor inactive control inhibitors have effects on dBest1currents. Our results demonstrate that dBest1 currents are regulated by phosphorylation via a CaMKII dependent mechanism.

Kv3.4 potassium channel-mediated electrosignaling controls cell cycle and survival of irradiated leukemia cells

Aberrant ion channel expression in the plasma membrane is characteristic for many tumor entities and has been attributed to neoplastic transformation, tumor progression, metastasis, and therapy resistance. The present study aimed to define the function of these "oncogenic" channels for radioresistance of leukemia cells. Chronic myeloid leukemia cells were irradiated (0-6Gy X ray), ion channel expression and activity, Ca(2+)- and protein signaling, cell cycle progression, and cell survival were assessed by quantitative reverse transcriptase-polymerase chain reaction, patch-clamp recording, fura-2 Ca(2+)-imaging, immunoblotting, flow cytometry, and clonogenic survival assays, respectively. Ionizing radiation-induced G2/M arrest was preceded by activation of Kv3.4-like voltage-gated potassium channels. Channel activation in turn resulted in enhanced Ca(2+) entry and subsequent activation of Ca(2+)/calmodulin-dependent kinase-II, and inactivation of the phosphatase cdc25B and the cyclin-dependent kinase cdc2. Accordingly, channel inhibition by tetraethylammonium and blood-depressing substance-1 and substance-2 or downregulation by RNA interference led to release from radiation-induced G2/M arrest, increased apoptosis, and decreased clonogenic survival. Together, these findings indicate the functional significance of voltage-gated K(+) channels for the radioresistance of myeloid leukemia cells.

Calmodulin-dependent kinase II regulates osteoblast differentiation through regulation of Osterix

Osterix (Osx), a zinc-finger transcription factor, is required for osteoblast differentiation and new bone formation during embryonic development. Calmodulin-dependent kinase II (CaMKII) acts as a key regulator of osteoblast differentiation. However, the precise molecular signaling mechanisms between Osterix and CaMKII are not known. In this study, we focused on the relationship between Osterix and CaMKII during osteoblast differentiation. We examined the role of the CaMKII pathway in the regulation of protein levels and its transcriptional activity on Osterix. We showed that CaMKII interacts with Osterix by increasing the protein levels and enhancing the transcriptional activity of Osterix. Conversely, CaMKII inhibitor KN-93 decreases the protein levels and increases the stability of Osterix. The siRNA-mediated knockdown of CaMKII decreased the protein levels and transcriptional activity of Osterix. These results suggest that Osterix is a novel target of CaMKII and the activity of Osterix can be modulated by a novel mechanism involving CaMKII during osteoblast differentiation.

Phosphorylation at Ser26 in the ATP-binding site of Ca2+/calmodulin-dependent kinase II as a mechanism for switching off the kinase activity

CaMKII (Ca2+/calmodulin-dependent kinase II) is a serine/threonine phosphotransferase that is capable of long-term retention of activity due to autophosphorylation at a specific threonine residue within each subunit of its oligomeric structure. The γ isoform of CaMKII is a significant regulator of vascular contractility. Here, we show that phosphorylation of CaMKII γ at Ser26, a residue located within the ATP-binding site, terminates the sustained activity of the enzyme. To test the physiological importance of phosphorylation at Ser26, we generated a phosphospecific Ser26 antibody and demonstrated an increase in Ser26 phosphorylation upon depolarization and contraction of blood vessels. To determine if the phosphorylation of Ser26 affects the kinase activity, we mutated Ser26 to alanine or aspartic acid. The S26D mutation mimicking the phosphorylated state of CaMKII causes a dramatic decrease in Thr287 autophosphorylation levels and greatly reduces the catalytic activity towards an exogenous substrate (autocamtide-3), whereas the S26A mutation has no effect. These data combined with molecular modelling indicate that a negative charge at Ser26 of CaMKII γ inhibits the catalytic activity of the enzyme towards its autophosphorylation site at Thr287 most probably by blocking ATP binding. We propose that Ser26 phosphorylation constitutes an important mechanism for switching off CaMKII activity.

Lateral thinking: CaMKII uncouples kainate receptors from mossy fibre synapses

Alteration of the efficacy of excitatory synaptic transmission between neurons is a critical element in the processes of learning, memory, and behaviour. Despite decades of research aimed at elucidating basic cellular mechanisms underlying synaptic plasticity, new pathways and permutations continue to be discovered. Carta et al (2013) now show that activation of the calcium/calmodulin dependent kinase II (CaMKII) induces an unusual postsynaptic form of long‐term depression (LTD) at the hippocampal mossy fibre synapse by promoting lateral diffusion of kainate receptors (KARs), a family of ionotropic glutamate receptors (iGluRs) that influence pyramidal neuron excitability. This report therefore reveals a new and mechanistically unique way of fine‐tuning synaptic plasticity at this central synapse in the hippocampus. Information transfer within the nervous system is regulated at the synaptic level by diverse cellular mechanisms. Synaptic efficacy is not static (i.e., it is ‘plastic’), and the capacity to adjust the strength of communication between neurons in a network has been shown to be a critical component of diverse aspects of brain function that include many forms of behavioural learning (Martin et al , 2000). The complex means by which neurons adjust their synaptic properties in response to changes in local and global activity in the central nervous system has been the subject of intensive investigation spanning multiple decades (Malenka and Bear, 2004; Feldman, 2009). Nonetheless, new mechanisms underlying plasticity of excitatory and inhibitory synaptic transmission continue to be elucidated; these can vary depending on the experimental parameters for induction of plasticity, the particular type of synapse under investigation, and even the prior history of activation at the synapse. Long‐term potentiation (LTP) and LTD …

CaMKII-dependent phosphorylation of GluK5 mediates plasticity of kainate receptors

Calmodulin-dependent kinase II (CaMKII) is key for long-term potentiation of synaptic AMPA receptors. Whether CaMKII is involved in activity-dependent plasticity of other ionotropic glutamate receptors is unknown. We show that repeated pairing of pre- and postsynaptic stimulation at hippocampal mossy fibre synapses induces long-term depression of kainate receptor (KAR)-mediated responses, which depends on Ca(2+) influx, activation of CaMKII, and on the GluK5 subunit of KARs. CaMKII phosphorylation of three residues in the C-terminal domain of GluK5 subunit markedly increases lateral mobility of KARs, possibly by decreasing the binding of GluK5 to PSD-95. CaMKII activation also promotes surface expression of KARs at extrasynaptic sites, but concomitantly decreases its synaptic content. Using a molecular replacement strategy, we demonstrate that the direct phosphorylation of GluK5 by CaMKII is necessary for KAR-LTD. We propose that CaMKII-dependent phosphorylation of GluK5 is responsible for synaptic depression by untrapping of KARs from the PSD and increased diffusion away from synaptic sites.

CASK regulates CaMKII autophosphorylation in neuronal growth, calcium signaling, and learning.

Calcium (Ca2+)/calmodulin (CaM)-dependent kinase II (CaMKII) activity plays a fundamental role in learning and memory. A key feature of CaMKII in memory formation is its ability to be regulated by autophosphorylation, which switches its activity on and off during synaptic plasticity. The synaptic scaffolding protein CASK (calcium (Ca2+)/calmodulin (CaM) associated serine kinase) is also important for learning and memory, as mutations in CASK result in intellectual disability and neurological defects in humans. We show that in Drosophila larvae, CASK interacts with CaMKII to control neuronal growth and calcium signaling. Furthermore, deletion of the CaMK-like and L27 domains of CASK (CASK β null) or expression of overactive CaMKII (T287D) produced similar effects on synaptic growth and Ca2+ signaling. CASK overexpression rescues the effects of CaMKII overactivity, consistent with the notion that CASK and CaMKII act in a common pathway that controls these neuronal processes. The reduction in Ca2+ signaling observed in the CASK β null mutant caused a decrease in vesicle trafficking at synapses. In addition, the decrease in Ca2+ signaling in CASK mutants was associated with an increase in Ether-à-go-go (EAG) potassium (K+) channel localization to synapses. Reducing EAG restored the decrease in Ca2+ signaling observed in CASK mutants to the level of wildtype, suggesting that CASK regulates Ca2+ signaling via EAG. CASK knockdown reduced both appetitive associative learning and odor evoked Ca2+ responses in Drosophila mushroom bodies, which are the learning centers of Drosophila. Expression of human CASK in Drosophila rescued the effect of CASK deletion on the activity state of CaMKII, suggesting that human CASK may also regulate CaMKII autophosphorylation.

Lamotrigine Increases Intracellular Calcium Levels and Camkii Activation in Mouse Dorsal Root Ganglion Neurons

Lamotrigine is a neuroprotective agent that is used clinically for the treatment of seizures and neuropathic pain. A significant volume of literature has reported that lamotrigine exerts analgesic effect by blocking Ca2+ channels. However, little is known regarding the effect of lamotrigine on the intracellular Ca2+ concentration ([Ca2+]i). The aim of this study was to determine whether lamotrigine modulates [Ca2+]i in sensory neurons. Lamotrigine-induced changes in [Ca2+]i were measured in mouse dorsal root ganglia (DRG) neurons using the Ca2+-sensitive fluorescent indicator Fluo 3-AM and a confocal laser scanning microscope. Ca2+/calmodulin-dependent kinase II (CaMKII) activation was assessed by fluorescence intensity using immunocytochemical procedures.Treatment with 1, 10, 30, or 100 μM lamotrigine transiently increased [Ca2+]i in DRG neurons in a dose-dependent manner. Treatment with 100 μM lamotrigine induced a significant (three-fold) increase in the Ca2+ peak in the presence or absence of extracellular Ca2+. The lamotrigine-induced Ca2+ increase was abolished or decreased by treatment with a specific PLC inhibitor (U73122), IP3R antagonist (xestospongin C), or RyR antagonist (dantrolene). In some cells, treatment with 100 μM lamotrigine caused a transient Ca2+ increase, and the Ca2+ levels quickly fell to below the basal Ca2+ level observed prior to lamotrigine application. The decrease in basal Ca2+ levels was blocked by treatment with a CaMKII inhibitor (KN93). Immunocytochemical analysis indicated that lamotrigine treatment increased the expression of phosphorylated CaMKII in DRG neurons.Treatment with lamotrigine increased [Ca2+]i apparently as a result of Ca2+ release from intracellular stores and CaMKII activity.Key words: Calcium, Ca2+/calmodulin-dependent kinase II, Dorsal Root Ganglia, Lamotrigine

WNT3A modulates chondrogenesis via canonical and non-canonical Wnt pathways in MSCs

The multilineage commitment of mesenchymal stem cells (MSCs) is controlled via unknown mechanisms. In this study, we investigated the regulation of the differentiation of MSCs into chondrocytes via the Wnt signaling pathway. Overexpression of WNT3A in MSCs activated both the canonical and non-canonical Wnt pathways, which were responsible for different WNT3A-induced outcomes. WNT3A promoted MSC proliferation via the ß-catenin-mediated canonical Wnt pathway, and inhibited chondrogenesis of MSCs via the calcium/calmodulin-dependent kinase II (CaMKII)-mediated non-canonical Wnt pathway. Interestingly, blockade of the canonical Wnt pathway by Dickkopf-related protein 1 exerted a synergistic effect on the inhibition of chondrogenesis of MSCs, while blockade of the non-canonical Wnt pathway by KN93 also exerted a synergistic effect on MSCs proliferation. These results suggest that the WNT3A-activated canonical and non-canonical pathways counteract each other in the setting of MSCs. This study provides evidence for the delicate regulation of the Wnt signaling cascade during chondrogenesis of MSCs, and suggests that genetic manipulation of the Wnt pathway may offer a powerful vehicle for modulating MSC differentiation in stem-cell-based cartilage repair.

Dual effect of the heart-targeting cytokine cardiotrophin-1 on glucose transport in cardiomyocytes

Cardiotrophin-1 (CT-1) is a heart-targeting cytokine that is increased in the metabolic syndrome due to overexpression in the adipocytes. The effects of CT-1 on cardiomyocyte substrate metabolism remain unknown. We therefore determined the effects of CT-1 on basal and stimulated glucose transport in cardiomyocytes exposed to a low dose (1nM) or a high dose (10nM). Dose–response curves for insulin showed that 1nM CT-1 reduced insulin responsiveness, while 10nM CT-1 increased insulin responsiveness. In either condition insulin sensitivity was unaffected. Similarly 1nM CT-1 reduced the stimulation of glucose transport in response to metabolic stress, induced by the mitochondrial poison oligomycin, while 10nM CT-1 increased this response. Reduction of stimulated glucose transport by 1nM CT-1 was associated with overexpression of SOCS-3, a protein known to hinder proximal insulin signaling, and increased phosphorylation of STAT5. In cardiomyocytes exposed to 1nM CT-1 there was also reduced phosphorylation of Akt and AS160 in response to insulin, and of AMPK in response to oligomycin. Insulin-stimulated glucose transport and signaling were restored by inhibition of STAT5 activity. On the other hand in cardiomyocytes exposed to 10nM CT-1 there was increased phosphorylation of the AS160 and Akt in response to insulin. Most importantly, basal and oligomycin-stimulated phosphorylation of AMPK was markedly increased in cardiomyocytes exposed to 10nM CT-1. The enhancement of basal and stimulated-glucose transport was abolished in cardiomyocytes treated with the calmodulin-dependent kinase II (CaMKII) inhibitor KN93, and so was AMPK phosphorylation. This suggests that activation of CaMKII mediates activation of AMPK by a high dose of CT-1 independently of metabolic stress. Our results point to a role for CT-1 in the regulation of myocardial glucose metabolism and implicate entirely separate mechanisms in the inhibitory or stimulatory effects of CT-1 on glucose transport at low or high concentrations respectively.

N-(Phenoxyalkyl)amides as MT1 and MT2 ligands: Antioxidant properties and inhibition of Ca2+/CaM-dependent kinase II

Recently a series of chiral N-(phenoxyalkyl)amides have been reported as potent MT1 and MT2 melatonergic ligands. Some of these compounds were selected and tested for their antioxidant properties by measuring their reducing effect against oxidation of 2′,7′-dichlorodihydrofluorescein (DCFH) in the DCFH-diacetate (DCFH-DA) assay. Among the tested compounds, N-[2-(3-methoxyphenoxy)propyl]butanamide displayed potent antioxidant activity that was stereoselective, the (R)-enantiomer performing as the eutomer. This compound displayed strong cytoprotective activity against H2O2-induced cytotoxicity resulting slightly more active than melatonin, and performed as Ca2+/calmodulin-dependent kinase II (CaMKII) inhibitor, too.

Lamotrigine increases intracellular Ca2+ levels and Ca2+/calmodulin‐dependent kinase II activation in mouse dorsal root ganglion neurones

Lamotrigine is a neuroprotective agent that is used clinically for the treatment of seizures and neuropathic pain. A significant volume of literature has reported that lamotrigine exerts analgesic effect by blocking Ca(2+) channels. However, little is known regarding the effect of lamotrigine on the intracellular Ca(2+) concentration ([Ca(2+)](i)). The aim of this study was to determine whether lamotrigine modulates [Ca(2+)](i) in sensory neurones. Lamotrigine-induced changes in [Ca(2+)](i) were measured in mouse dorsal root ganglion (DRG) neurones using the Ca(2+)-sensitive fluorescent indicator Fluo 3-AM and a confocal laser scanning microscope. Ca(2+)/calmodulin-dependent kinase II (CaMKII) activation was assessed by the fluorescence intensity using immunocytochemical procedures. Treatment with 1, 10, 30 or 100 μM lamotrigine transiently increased [Ca(2+)](i) in DRG neurones in a dose-dependent manner. Treatment with 100 μM lamotrigine induced a significant (threefold) increase in the Ca(2+) peak in the presence or absence of extracellular Ca(2+). The lamotrigine-induced Ca(2+) increase was abolished or decreased by the treatment with a specific PLC inhibitor (U73122), IP3R antagonist (xestospongin C) or RyR antagonist (dantrolene). In some cells, treatment with 100 μM lamotrigine caused a transient Ca(2+) increase, and the Ca(2+) levels quickly fell to below the basal Ca(2+) level observed prior to lamotrigine application. The decrease in basal Ca(2+) levels was blocked by the treatment with a CaMKII inhibitor (KN93). Immunocytochemical analysis indicated that lamotrigine treatment increased the expression of phosphorylated CaMKII in DRG neurones. Treatment with lamotrigine increased [Ca(2+)](i) apparently as a result of Ca(2+) release from intracellular stores and CaMKII activity.

Abstract 9785: The Contribution of CaMKII and PKA to the Development of an Increased RyR-dependent SR Ca Leak in Human Cardiac Pathology

The mechanisms of a progressive ryanodine receptor type 2 (RyR2) dysregulation in the transition from human cardiac hypertrophy (Hy) to heart failure (HF) are still unclear. Inhibition of Ca 2+ /calmodulin-dependent kinase II (CaMKII) reduces the sarcoplasmic reticulum (SR) Ca 2+- leak and improve contractility in human HF. However, the relative contribution of CaMKII and protein kinase A are still not clear. Western blot experiments from tissue samples (n=12) of patients with afterload-induced Hy and preserved ejection fraction (EF>50%) revealed an increase in RyR2 expression by 82±22% ( P =0.05) compared to healthy controls (n=5). The phosphorylation status of RyR2 at S2809 and S2815 was not altered. In human end-stage HF (n=8), a pronounced hyperphosphorylation of RyR2 could be detected at the CaMKII-dependent site S2815 (by 311±72%, P <0.05) whereas the PKA-dependent site S2809 lacked differential regulation. The comparison of HF patients with and without β-blocker medication (n=7 vs. 4) did not reveal any differences as to RyR2 phosphorylation status. When isolated myocytes were paced at 1 Hz and scanned for diastolic Ca 2+ sparks (confocal microscopy, Fluo 3) a higher Ca 2+ -spark frequency (by 82±26% P <0.05) was detected in HF (n=93) compared to Hy (n=94). Interestingly, PKA inhibition with H89 (5 μM) only accomplished a decrease of SR Ca 2+ -leak in Hy (by 59±13%, P =0.05, n=85 vs. 115) but not in HF (n=85 vs. 74). Stimulated Ca 2+ transients (0.5 Hz, n=31 vs. 23), SR Ca 2+ content (caffeine, n=23 vs. 22) and fractional SR Ca 2+ release (n=21 vs. 15) were not changed by H89 treatment in HF (epifluorescence microscopy, Fura 2). CaMKII inhibition by AIP (1 μM), in contrast, could significantly reduce SR Ca 2+ leak in Hy (by 63±14%, P <0.05, n=93 vs. 103) as well as in HF. Thus, in human Hy with preserved EF both kinases seem to functionally regulate RyR2 gating. In end-stage human HF, however, the diastolic SR Ca 2+ leak seems to be induced by CaMKII rendering an inhibition of CaMKII a promising pharmacologic toehold.

Regulatory roles of the NMDA receptor GluN3A subunit in locomotion, pain perception and cognitive functions in adult mice

Since the discovery of the glutamate NMDA receptor subunit 3A (GluN3A), the functional role of this unique inhibitory subunit has been largely obscure. GluN3A expression is high in the neonatal brain but declines to a low level in the adult brain; it is thus commonly believed that GluN3A does not have a major functional impact in adulthood. Using wild-type (WT) and GluN3A knockout (KO) mice, we show here that deletion of GluN3A affected multiple behavioural functions in adult animals. GluN3A KO mice showed impaired locomotor activity on a variety of motor function tests, and increased sensitivity to acute and sub-acute inflammatory pain. GluN3A KO mice also showed enhanced recognition and spatial learning and memory functions. Hippocampal slices from juvenile and adult GluN3A KO mice showed greater long-term potentiation (LTP) compared with WT slices. GluN3A deletion resulted in increased expression of Ca(2+)/calmodulin-dependent kinase II (CaMKII) in the forebrain, and the phosphorylated CaMKII level upon LTP induction was significantly higher in the GluN3A KO hippocampus compared with WT controls. CaMKII inhibition abrogated the enhanced LTP in GluN3A KO slices. These data reveal for the first time that the presence of GluN3A may have profound impacts on several functional/behavioural activities in adult animals, and could be a therapeutic target for neurological disorders associated with NMDA receptor functions.

Modelling Ca2+ -dependent proteins in the spine - challenges and solutions

Background / Purpose: Modelling post-synaptic proteins poses three technical problems: small absolute molecule numbers, large numbers of possible states, and the complex geometry of the spine, which is not a well-mixed compartment. Computational approaches are needed that solve all three of these problems. Main conclusion: Stochastic simulation methods can be used for systems with small molecule numbers, agent-based methods to represent multi-state molecules, and spatial methods to simulate events in complex geometries. We used the agent-based spatial stochastic simulator MCell to model the Ca2+-dependent activation of calmodulin and Ca2+/calmodulin-dependent kinase II (CaMKII) in the spine. Next steps: Next steps will include the extension of our model to include more interaction partners, and to represent some of the regulation events in more detail.