Calmodulin-dependent Protein Kinase II Binding Research Articles

PFOS Elicits Cytotoxicity in Neuron Through Astrocyte-Derived CaMKII-DLG1 Signaling In Vitro Rat Hippocampal Model.

Both epidemiological investigation and animal experiments demonstrated that pre-/postnatal exposure to perfluorooctane sulfonic acid (PFOS) could induce neurodevelopmental disorders. Previous studies showed that astrocyte was involved in PFOS-induced neurotoxicity, while little information is available. In the present study, the role of astrocyte-derived calmodulin-dependent protein kinase II (CaMKII)-phosphorylated discs large homolog 1 (DLG1) signaling in PFOS eliciting cytotoxicity in neuron was explored with primary cultured hippocampal astrocyte and neuron. The application of PFOS showed a decreased cell viability, synapse length and glutamate transporter 1 (GLT-1) expression, but an increased CaMKII, DLG1 and cyclic AMP response element binding protein (CREB) expression in primary cultured astrocyte. With 2-(2-hydroxyethylamino)-6-aminohexylcarbamic acid tert-butyl ester-9-isopropylpurine (CK59), the CaMKII inhibitor, the disturbed cell viability and molecules induced by PFOS could be alleviated (CREB expression was excluded) in astrocytes. The cytotoxic effect of neuron exposed to astrocyte conditional medium collected from PFOS (PFOS-ACM) pretreated with CK59 was also decreased. These results indicated that PFOS mediated GLT-1 expression through astrocyte-derived CaMKII-DLG signaling, which might be associated with injuries on neurons. The present study gave an insight in further exploration of mechanism in PFOS-induced neurotoxicity.

CaMKII T286 phosphorylation has distinct essential functions in three forms of long-term plasticity

The Ca2+/calmodulin-dependent protein kinase II (CaMKII) mediates long-term potentiation or depression (LTP or LTD) after distinct stimuli of hippocampal NMDA-type glutamate receptors (NMDARs). NMDAR-dependent LTD prevails in juvenile mice, but a mechanistically different form of LTD can be readily induced in adults by instead stimulating metabotropic glutamate receptors (mGluRs). However, the role that CaMKII plays in the mGluR-dependent form of LTD is not clear. Here we show that mGluR-dependent LTD also requires CaMKII and its T286 autophosphorylation (pT286), which induces Ca2+-independent autonomous kinase activity. In addition, we compared the role of pT286 among three forms of long-term plasticity (NMDAR-dependent LTP and LTD, and mGluR-dependent LTD) using simultaneous live imaging of endogenous CaMKII together with synaptic marker proteins. We determined that after LTP stimuli, pT286 autophosphorylation accelerated CaMKII movement to excitatory synapses. After NMDAR-LTD stimuli, pT286 was strictly required for any movement to inhibitory synapses. Similar to NMDAR-LTD, we found the mGluR-LTD stimuli did not induce CaMKII movement to excitatory synapses. However, in contrast to NMDAR-LTD, we demonstrate that the mGluR-LTD did not involve CaMKII movement to inhibitory synapses and did not require additional T305/306 autophosphorylation. Thus, despite its prominent role in LTP, we conclude that CaMKII T286 autophosphorylation is also required for both major forms of hippocampal LTD, albeit with differential requirements for the heterosynaptic communication of excitatory signals to inhibitory synapses.

Elucidating the Mechanisms of CaMKII‐CaMKAP Interactions

Dynamic changes of intracellular calcium ion (Ca2+) concentrations are critical signaling mechanisms in all cells and organisms, and Ca2+ oscillations must be precisely decoded by intracellular signaling molecules, such as the ubiquitous Ca2+/calmodulin‐dependent protein kinase II (CaMKII). Four mammalian CaMKII genes encode diverse alternative mRNA splice variants, which are thought to be targeted to different subcellular compartments to regulate numerous cell responses (exocytosis, gene expression, cell cycle). Our lab hypothesized that the localization, activity, and biological functions of CaMKII are precisely modulated by CaMKII‐associated proteins (CaMKAPs), and we have detected over 100 CaMKAPs associated with brain CaMKII holoenzymes. Some CaMKII‐CaMKAP interactions are critical for normal physiological function. CaMKIIδ binding to the L‐type Ca2+ channel (LTCC) b2a subunit is essential for cardiac function1, and binding of neuronal CaMKII to the GluN2B subunit of the NMDA‐type glutamate receptor is necessary for synaptic plasticity2. While b2a, GluN2B and most other CaMKAPs preferentially interact with activated CaMKII, the exact molecular mechanisms for these interactions, and their physiological roles, have yet to be determined. Several residues in the catalytic domain of CaMKII have been implicated in CaMKAP binding. Previous mutagenesis studies from our lab indicated that mutation of valine 102 (V102) to glutamate disrupts CaMKII binding to LTCC α1 subunits, but not to b2a, GluN2B, or densin, and also disrupts neuronal excitation‐transcription coupling3. Additionally, a naturally‐occurring CaMKIIα Glu183Val mutation linked to autism spectrum disorder and intellectual disability disrupted interactions with all CaMKAPs tested4. To further probe the molecular basis of CaMKII‐CaMKAP interactions, we are exploiting available structural information to design additional CaMKII catalytic domain mutations, and then test their effect on CaMKII‐CaMKAP interactions, as well as CaMKII activity toward multiple substrates. We are also examining the impact of additional human CaMKII mutations. Initial studies indicate that some CaMKII mutations differentially affect binding to various CaMKAPs, CaMKII autophosphorylation, and phosphorylation of a representative substrate, the GluA1 subunit of AMPA‐type glutamate receptors. Our ongoing studies are further exploring the impact of these CaMKII mutations. Expanding our knowledge of mechanisms underlying CaMKII‐CaMKAP interactions is required to better understand CaMKII biology, the role of CaMKAPs, and the molecular basis of Ca2+signaling. Koval et al., 2010. PNAS 107:4996. Halt et al., 2012. EMBO J. 31:1203. Wang et al., 2017. J Biol Chem 292:17324. Stephenson et al., 2017. J Neurosci 37:2216.

Convergent NMDA receptor\u2014Pannexin1 signaling pathways regulate the interaction of CaMKII with Connexin-36

Ca2+/calmodulin-dependent protein kinase II (CaMKII) binding and phosphorylation of mammalian connexin-36 (Cx36) potentiate electrical coupling. To explain the molecular mechanism of how Cx36 modifies plasticity at gap junctions, we investigated the roles of ionotropic N-methyl-D-aspartate receptors and pannexin1 (Panx1) channels in regulating Cx36 binding to CaMKII. Pharmacological interference and site-directed mutagenesis of protein interaction sites shows that NMDA receptor activation opens Cx36 channels, causing the Cx36- CaMKII binding complex to adopt a compact conformation. Ectopic Panx1 expression in a Panx1 knock-down cell line is required to restore CaMKII mediated opening of Cx36. Furthermore, blocking of Src-family kinase activation of Panx1 is sufficient to prevent the opening of Cx36 channels. Our research demonstrates that the efficacy of Cx36 channels requires convergent calcium-dependent signaling processes in which activation of ionotropic N-methyl-D-aspartate receptor, Src-family kinase, and Pannexin1 open Cx36. Our results add to the best of our knowledge a new twist to mounting evidence for molecular communication between these core components of electrical and chemical synapses.

κ Opioid Receptor Agonist Inhibits Myocardial Injury in Heart Failure Rats through Activating Nrf2/HO-1 Pathway and Regulating Ca2+-SERCA2a.

Objectives We aimed to observe the protective effect of κ opioid receptor (κ-OR) agonist on myocardial injury in heart failure (HF) rats and its effect on Ca2+-SERCA2a and to explore the regulatory mechanism with the Nrf2/HO-1 signaling pathway. Methods 50 Sprague-Dawley rats were randomly divided into the following groups: the sham operation group (sham group), HF model group (HF group), HF+κ-OR agonist U50488 group (HU group), HF+U50488H+novel calmodulin-dependent protein kinase II (CaMKII) agonist (oleic acid) (HUO group), and HF+U50488H+Nrf2 inhibitor (HUM group). The HF rat's model was established through surgical ligation of the left anterior descending coronary artery and the exhausting swimming exercise. After that, rat's cardiac function was monitored by echocardiography. HE and MASSON staining was used to detect the myocardial injury, and TUNEL staining was used to detect the myocardial apoptosis. ELISA was performed to detect the biomarkers of oxidative stress. Moreover, the distribution of reactive oxygen species (ROS) and Nrf2 was detected under immunofluorescence. The expression of sarco/endoplasmic reticulum calcium (Ca2+) ATPase (SERCA) 2a, calmodulin, endoplasmic reticulum stress- (ERS-) related proteins, and Nrf2/HO-1 signaling pathway-related proteins were detected by Western Blotting. Results κ-OR agonist U50488H can significantly enhance rat's cardiac function, reduce the injury and apoptosis of myocardial cells, and alleviate endoplasmic reticulum stress injury in HF rats via upregulating the SERCA2a expression and inhibiting the Ca2+ influx. Furthermore, U50488H could also inhibit the phosphorylation of CaMKII and cAMP-response element binding protein (CREB). Additionally, administration of CaMKII-specific agonist could partially block the therapeutic effect of κ-OR agonist on the myocardium of HF rats. Interestingly, the antagonist of Nrf2 could also significantly reverse the therapeutic effect of κ-OR agonist. Therefore, these results suggested that the effect of U50488H on HF rats is dependent on regulating CaMKII phosphorylation and activating the Nrf2/HO-1 pathway. Conclusion κ-OR agonists U50488H can improve ERS in cardiomyocytes and relieve myocardial injury in HF rats through activating the Nrf2/HO-1 pathway and regulating Ca2+-SERCA2a to inhibit Ca2+ influx.

Charged Side Chain Mutations of CaMKII Peptide Alter Binding Affinity for CaM through Creation of Non-local Alternate Binding Contacts

Calmodulin (CaM) trapping is a phenomenon where the active and inactive states of calmodulin dependent protein kinase II (CaMKII) produce drastically different affinities for Ca2+ saturated CaM. The states of CaMKII are paramount in the scheme of calcium ion signaling, which is an essential biological function whose underlying mechanism is largely unknown. Experimentally, a set of peptides modeled after CaMKII's binding domain (293-312) were created through systematic mutations of charged residues to mimic the two distinct affinity states for CaM and probe the mechanisms responsible for the observed change in kinetics. Although a model was successfully created, the observed interactions could not be explained through current protein interaction models or electrostatic steering effects. We investigate the dynamics of this experiment through the use of all atom simulations, choosing three of the mutant peptides with a length of 20 amino acids. We refer to these peptides by the 296-298 residues, specifically RRK (wildtype), RAK (1-residue mutation) and AAA (3-residue mutation), and validate our simulation findings through comparison with circular dichroism (CD) data. We demonstrate that the large side chains present in the first 6 residues of each peptide interact with each other, and that the mutation of charged residues produce global changes in sidechain conformations and disordered regions. The specific ensemble of secondary structure/rotamer conformations present in RRK, that are not observed in AAA, are essential for high affinity binding between CaMKII and CaM.

DAPK1 Mediates LTD by Making CaMKII/GluN2B Binding LTP Specific

The death-associated protein kinase 1 (DAPK1) is a potent mediator of neuronal cell death. Here, we find that DAPK1 also functions in synaptic plasticity by regulating the Ca2+/calmodulin (CaM)-dependent protein kinase II (CaMKII). CaMKII and T286 autophosphorylation are required for both long-term potentiation (LTP) and depression (LTD), two opposing forms of synaptic plasticity underlying learning, memory, and cognition. T286-autophosphorylation induces CaMKII binding to the NMDA receptor (NMDAR) subunit GluN2B, which mediates CaMKII synaptic accumulation during LTP. We find that the LTP specificity of CaMKII synaptic accumulation is due to its LTD-specific suppression by calcineurin (CaN)-dependent DAPK1 activation, which in turn blocks CaMKII binding to GluN2B. This suppression is enabled by competitive DAPK1 versus CaMKII binding to GluN2B. Negative regulation of DAPK1/GluN2B binding by Ca2+/CaM results in synaptic DAPK1 removal during LTP but retention during LTD. A pharmacogenetic approach showed that suppressionof CaMKII/GluN2B binding is a DAPK1 function required for LTD.

Multiple CaMKII Binding Modes to the Actin Cytoskeleton Revealed by Single-Molecule Imaging

Localization of the Ca2+/calmodulin-dependent protein kinase II (CaMKII) to dendritic spine synapses is determined in part by the actin cytoskeleton. We determined binding of GFP-tagged CaMKII to tag-RFP-labeled actin cytoskeleton within live cells using total internal reflection fluorescence microscopy and single-molecule tracking. Stepwise photobleaching showed that CaMKII formed oligomeric complexes. Photoactivation experiments demonstrated that diffusion out of the evanescent field determined the track lifetimes. Latrunculin treatment triggered a coupled loss of actin stress fibers and the colocalized, long-lived CaMKII tracks. The CaMKIIα (α) isoform, which was previously thought to lack F-actin interactions, also showed binding, but this was threefold weaker than that observed for CaMKIIβ (β). The βE′ splice variant bound more weakly than α, showing that binding by β depends critically on the interdomain linker. The mutations βT287D and αT286D, which mimic autophosphorylation states, also abolished F-actin binding. Autophosphorylation triggers autonomous CaMKII activity, but does not impair GluN2B binding, another important synaptic protein interaction of CaMKII. The CaMKII inhibitor tatCN21 or CaMKII mutations that inhibit GluN2B association by blocking binding of ATP (βK43R and αK42M) or Ca2+/calmodulin (βA303R) had no effect on the interaction with F-actin. These results provide the first rationale for the reduced synaptic spine localization of the αT286D mutant, indicating that transient F-actin binding contributes to the synaptic localization of the CaMKIIα isoform. The track lifetime distributions had a stretched exponential form consistent with a heterogeneously diffusing population. This heterogeneity suggests that CaMKII adopts different F-actin binding modes, which is most easily rationalized by multiple subunit contacts between the CaMKII dodecamer and the F-actin cytoskeleton that stabilize the initial weak (micromolar) monovalent interaction.

Nanoparticle as signaling protein mimic: robust structural and functional modulation of CaMKII upon specific binding to fullerene C60 nanocrystals.

In a biological environment, nanoparticles encounter and interact with thousands of proteins, forming a protein corona on the surface of the nanoparticles, but these interactions are oftentimes perceived as nonspecific protein adsorption, with protein unfolding and deactivation as the most likely consequences. The potential of a nanoparticle-protein interaction to mimic a protein-protein interaction in a cellular signaling process, characterized by stringent binding specificity and robust functional modulation for the interacting protein, has not been adequately demonstrated. Here, we show that water-suspended fullerene C60 nanocrystals (nano-C60) interact with and modulate the function of the Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), a multimeric intracellular serine/threonine kinase central to Ca(2+) signal transduction, in a fashion that rivals the well-documented interaction between the NMDA (N-methyl-d-aspartate) receptor subunit NR2B protein and CaMKII. The stable high-affinity binding of CaMKII to distinct sites on nano-C60, mediated by amino acid residues D246 and K250 within the catalytic domain of CaMKIIα, but not the nonspecific adsorption of CaMKII to diamond nanoparticles, leads to functional consequences reminiscent of the NR2B-CaMKII interaction, including generation of autonomous CaMKII activity after Ca(2+) withdrawal, calmodulin trapping and CaMKII translocation to postsynaptic sites. Our results underscore the critical importance of specific interactions between nanoparticles and cellular signaling proteins, and the ability of nano-C60 to sustain the autonomous kinase activity of CaMKII may have significant implications for both the biosafety and the potential therapeutic applications of fullerene C60.

CaMKII binding to GluN2B is differentially affected by macromolecular crowding reagents.

Binding of the Ca2+/calmodulin(CaM)-dependent protein kinase II (CaMKII) to the NMDA-type glutamate receptor (NMDAR) subunit GluN2B controls long-term potentiation (LTP), a form of synaptic plasticity thought to underlie learning and memory. Regulation of this interaction is well-studied biochemically, but not under conditions that mimic the macromolecular crowding found within cells. Notably, previous molecular crowding experiments with lysozyme indicated an effect on the CaMKII holoenzyme conformation. Here, we found that the effect of molecular crowding on Ca2+/CaM-induced CaMKII binding to immobilized GluN2B in vitro depended on the specific crowding reagent. While binding was reduced by lysozyme, it was enhanced by BSA. The ATP content in the BSA preparation caused CaMKII autophosphorylation at T286 during the binding reaction; however, enhanced binding was also observed when autophosphorylation was blocked. Importantly, the positive regulation by nucleotide and BSA (as well as other macromolecular crowding reagents) did not alleviate the requirement for CaMKII stimulation to induce GluN2B binding. The differential effect of lysozyme (14 kDa) and BSA (66 kDa) was not due to size difference, as both dextran-10 and dextran-70 enhanced binding. By contrast, crowding with immunoglobulin G (IgG) reduced binding. Notably, lysozyme and IgG but not BSA directly bound to Ca2+/CaM in an overlay assay, suggesting a competition of lysozyme and IgG with the Ca2+/CaM-stimulus that induces CaMKII/GluN2B binding. However, lysozyme negatively regulated binding even when it was instead induced by CaMKII T286 phosphorylation. Alternative modes of competition would be with CaMKII or GluN2B, and the negative effects of lysozyme and IgG indeed also correlated with specific or non-specific binding to the immobilized GluN2B. Thus, the effect of any specific crowding reagent can differ, depending on its additional direct effects on CaMKII/GluN2B binding. However, the results of this study also indicate that, in principle, macromolecular crowding enhances CaMKII binding to GluN2B.

Enzymatic Activity of CaMKII Is Not Required for Its Interaction with the Glutamate Receptor Subunit GluN2B

Binding of the Ca²⁺/calmodulin (CaM)-dependent protein kinase II (CaMKII) to the NMDA-type glutamate receptor subunit GluN2B is an important control mechanism for the regulation of synaptic strength. CaMKII binding to GluN2B and CaMKII translocation to synapses are induced by an initial Ca²⁺/CaM stimulus, which also activates the kinase. Indeed, several mechanistically different CaMKII inhibitors [tatCN21 and KN-93 (N-[2-[[[3-(4-chlorophenyl)-2-propenyl]methylamino]methyl]phenyl]-N-(2-hydroxyethyl)-4-methoxybenzenesulphonamide)] and inactivating mutations (K42M, A302R, and T305/T306D) impair this interaction, suggesting that it requires CaMKII enzymatic activity. However, this study shows that two general kinase inhibitors, H7 [1-(5-isoquinolinylsulfonyl)-2-methylpiperazine] and staurosporine (Sta), which inhibit CaMKII activity by yet another mechanism, did not interfere with GluN2B binding in vitro or within cells. In contrast to a previous report, we found that Sta, like H7, inhibited CaMKII in an ATP-competitive manner. Nucleotide binding significantly enhances CaMKII/GluN2B binding in vitro, but the nucleotide competition by H7 or Sta did not prevent this effect and instead even mimicked it. H7 (700 µM) and Sta (2 µM) efficiently blocked enzymatic activity of CaMKII, both in vitro and within cells. However, neither H7 nor Sta prevented Ca²⁺-induced translocation of CaMKII to GluN2B in heterologous cells or to synapses in hippocampal neurons. Thus, activity of CaMKII (or of any other kinase inhibited by H7 or Sta) is not required for stimulation-induced GluN2B-binding or synaptic translocation of CaMKII, despite previous indication to the contrary. This shows that results with inhibitors and inhibiting mutants can be caused by structural effects independent from catalytic activity, and that detailed understanding of the mechanisms is required for their interpretation.

CaMKII Does It Again

Although the recent identification of the mitochondrial Ca2+ uniporter (MCU) has resolved a long-standing mystery as to how Ca2+ freely enters the mitochondria, it has also evoked additional questions such as its mode of regulation and the identity of other associated factors. In an article recently published in Nature , Joiner et al1 provide data demonstrating that in the heart, matrix-localized Ca2+/calmodulin-dependent protein kinase II (CaMKII) can upregulate MCU activity in a manner requiring phosphorylation of the channel N terminus. They showed that inhibition of CaMKII-dependent MCU activity protected the heart from ischemic injury by presumably reducing Ca2+ influx and desensitizing the mitochondrial permeability transition pore (MPTP) to opening. Although these results demonstrate convincingly that CaMKII plays an important role in MCU regulation and subsequent response to cardiac injury, several questions remain unanswered. The ability of mitochondria to take up and sequester Ca2+ plays an important role in the buffering of cytosolic Ca2+, regulation of ATP production via the citric acid cycle, and regulation of apoptotic and necrotic cell death pathways.2 Although mitochondrial Ca2+ uptake was first described in the 1960s3 and the electrophysiological properties of the MCU were reported in 2004,4 it was not until 2011 that 2 articles were published revealing the genetic identity of the MCU.5,6 This pioneering work has initiated a search for additional members of the MCU complex (such as MICU17 and the recently discovered MCUR1),8 as well as an attempt to understand how MCU-mediated Ca2+ influx participates in the regulation of whole-cell Ca2+ signaling and whether well-described pathways that regulate other Ca2+ handling processes can similarly modulate MCU-dependent Ca2+ uptake. In the featured article, Joiner et al1 show that CaMKII serves …

Ca2+-independent Activation of Ca2+/Calmodulin-dependent Protein Kinase II Bound to the C-terminal Domain of CaV2.1 Calcium Channels

Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) forms a major component of the postsynaptic density where its functions in synaptic plasticity are well established, but its presynaptic actions are poorly defined. Here we show that CaMKII binds directly to the C-terminal domain of Ca(V)2.1 channels. Binding is enhanced by autophosphorylation, and the kinase-channel signaling complex persists after dephosphorylation and removal of the Ca(2+)/CaM stimulus. Autophosphorylated CaMKII can bind the Ca(V)2.1 channel and synapsin-1 simultaneously. CaMKII binding to Ca(V)2.1 channels induces Ca(2+)-independent activity of the kinase, which phosphorylates the enzyme itself as well as the neuronal substrate synapsin-1. Facilitation and inactivation of Ca(V)2.1 channels by binding of Ca(2+)/CaM mediates short term synaptic plasticity in transfected superior cervical ganglion neurons, and these regulatory effects are prevented by a competing peptide and the endogenous brain inhibitor CaMKIIN, which blocks binding of CaMKII to Ca(V)2.1 channels. These results define the functional properties of a signaling complex of CaMKII and Ca(V)2.1 channels in which both binding partners are persistently activated by their association, and they further suggest that this complex is important in presynaptic terminals in regulating protein phosphorylation and short term synaptic plasticity.

Wenxin-Keli Regulates the Calcium/Calmodulin-Dependent Protein Kinase II Signal Transduction Pathway and Inhibits Cardiac Arrhythmia in Rats with Myocardial Infarction

Wenxin-Keli (WXKL) is a Chinese herbal compound reported to be of benefit in the treatment of cardiac arrhythmia, cardiac inflammation, and heart failure. Amiodarone is a noncompetitive inhibitor of the α- and β-adrenergic receptors and prevents calcium influx in the slow-response cells of the sinoatrial and atrioventricular nodes. Overexpression of Ca2+/calmodulin-dependent protein kinase II (CaMKII) in transgenic mice results in heart failure and arrhythmias. We hypothesised that administration of WXKL and amiodarone can reduce the incidence of arrhythmias by regulating CaMKII signal transduction. A total of 100 healthy Sprague Dawley rats were used in the study. The rats were randomly divided into four groups (a sham group, a myocardial infarction (MI) group, a WXKL-treated group, and an amiodarone-treated group). A myocardial infarction model was established in these rats by ligating the left anterior descending coronary artery for 4 weeks. Western blotting was used to assess CaMKII, p-CaMKII (Thr-286), PLB, p-PLB (Thr-17), RYR2, and FK binding protein 12.6 (FKBP12.6) levels. The Ca2+ content in the sarcoplasmic reticulum (SR) and the calcium transient amplitude were studied by confocal imaging using the fluorescent indicator Fura-4. In conclusion, WXKL may inhibit heart failure and cardiac arrhythmias by regulating the CaMKII signal transduction pathway similar to amiodarone.

Calmodulin dependent protein kinase increases conductance at gap junctions formed by the neuronal gap junction protein connexin36

The major neuronal gap junction protein connexin36 (Cx36) exhibits the remarkable property of "run-up", in which junctional conductance typically increases by 10-fold or more within 5-10min following cell break-in with patch pipettes. Such conductance "run-up" is a unique property of Cx36, as it has not been seen in cell pairs expressing other connexins. Because of the recent observation describing CaMKII binding and phosphorylation sites in Cx36 and evidence that calmodulin dependent protein kinase II (CaMKII) may potentiate electrical coupling in neurons of teleosts, we have explored whether CaMKII activates mammalian Cx36. Consistent with this hypothesis, certain Cx36 mutants lacking the CaMKII binding and phosphorylation sites or wild type Cx36 treated with certain cognate peptides corresponding to binding or phosphorylation sites blocked or strongly attenuated run-up of junctional conductance. Likewise, KN-93, an inhibitor of CaMKII, blocked run-up, as did a membrane permeable peptide corresponding to the CaMKII autoinhibitory domain. Furthermore, run-up was blocked by phosphatase delivered within the pipette and not affected by treatment with the phosphatase inhibitor okadaic acid. These results imply that phosphorylation by CaMKII strengthens junctional currents of Cx36 channels, thereby conferring functional plasticity on electrical synapses formed of this protein.

Ca2+/Calmodulin-dependent Protein Kinase II (CaMKII) Regulates Cardiac Sodium Channel NaV1.5 Gating by Multiple Phosphorylation Sites

The cardiac Na(+) channel Na(V)1.5 current (I(Na)) is critical to cardiac excitability, and altered I(Na) gating has been implicated in genetic and acquired arrhythmias. Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is up-regulated in heart failure and has been shown to cause I(Na) gating changes that mimic those induced by a point mutation in humans that is associated with combined long QT and Brugada syndromes. We sought to identify the site(s) on Na(V)1.5 that mediate(s) the CaMKII-induced alterations in I(Na) gating. We analyzed both CaMKII binding and CaMKII-dependent phosphorylation of the intracellularly accessible regions of Na(V)1.5 using a series of GST fusion constructs, immobilized peptide arrays, and soluble peptides. A stable interaction between δ(C)-CaMKII and the intracellular loop between domains 1 and 2 of Na(V)1.5 was observed. This region was also phosphorylated by δ(C)-CaMKII, specifically at the Ser-516 and Thr-594 sites. Wild-type (WT) and phosphomutant hNa(V)1.5 were co-expressed with GFP-δ(C)-CaMKII in HEK293 cells, and I(Na) was recorded. As observed in myocytes, CaMKII shifted WT I(Na) availability to a more negative membrane potential and enhanced accumulation of I(Na) into an intermediate inactivated state, but these effects were abolished by mutating either of these sites to non-phosphorylatable Ala residues. Mutation of these sites to phosphomimetic Glu residues negatively shifted I(Na) availability without the need for CaMKII. CaMKII-dependent phosphorylation of Na(V)1.5 at multiple sites (including Thr-594 and Ser-516) appears to be required to evoke loss-of-function changes in gating that could contribute to acquired Brugada syndrome-like effects in heart failure.

Substrate-selective and Calcium-independent Activation of CaMKII by α-Actinin

Protein-protein interactions are thought to modulate the efficiency and specificity of Ca(2+)/calmodulin (CaM)-dependent protein kinase II (CaMKII) signaling in specific subcellular compartments. Here we show that the F-actin-binding protein α-actinin targets CaMKIIα to F-actin in cells by binding to the CaMKII regulatory domain, mimicking CaM. The interaction with α-actinin is blocked by CaMKII autophosphorylation at Thr-306, but not by autophosphorylation at Thr-305, whereas autophosphorylation at either site blocks Ca(2+)/CaM binding. The binding of α-actinin to CaMKII is Ca(2+)-independent and activates the phosphorylation of a subset of substrates in vitro. In intact cells, α-actinin selectively stabilizes CaMKII association with GluN2B-containing glutamate receptors and enhances phosphorylation of Ser-1303 in GluN2B, but inhibits CaMKII phosphorylation of Ser-831 in glutamate receptor GluA1 subunits by competing for activation by Ca(2+)/CaM. These data show that Ca(2+)-independent binding of α-actinin to CaMKII differentially modulates the phosphorylation of physiological targets that play key roles in long-term synaptic plasticity.

Calmodulin Kinase II Constitutively Binds, Phosphorylates, and Inhibits Brush Border Na+/H+ Exchanger 3 (NHE3) by a NHERF2 Protein-dependent Process

The epithelial brush border (BB) Na(+)/H(+) exchanger 3 (NHE3) accounts for most renal and intestinal Na(+) absorption. Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) inhibits NHE3 activity under basal conditions in intact intestine, acting in the BB, but the mechanism is unclear. We now demonstrate that in both PS120 fibroblasts and polarized Caco-2BBe cells expressing NHE3, CaMKII inhibits basal NHE3 activity, because the CaMKII-specific inhibitors KN-93 and KN-62 stimulate NHE3 activity. This inhibition requires NHERF2. CaMKIIγ associates with NHE3 between aa 586 and 605 in the NHE3 C terminus in a Ca(2+)-dependent manner, with less association when Ca(2+) is increased. CaMKII inhibits NHE3 by an effect on its turnover number, not changing surface expression. Back phosphorylation demonstrated that NHE3 is phosphorylated by CaMKII under basal conditions. This overall phosphorylation of NHE3 is not affected by the presence of NHERF2. Amino acids downstream of NHE3 aa 690 are required for CaMKII to inhibit basal NHE3 activity, and mutations of the three putative CaMKII phosphorylation sites downstream of aa 690 each prevented KN-93 stimulation of NHE3 activity. These studies demonstrate that CaMKIIγ is a novel NHE3-binding protein, and this association is reduced by elevated Ca(2+). CaMKII inhibits basal NHE3 activity associated with phosphorylation of NHE3 by effects requiring aa downstream of NHE3 aa 690 and of the CaMKII-binding site on NHE3. CaMKII binding to and phosphorylation of the NHE3 C terminus are parts of the physiologic regulation of NHE3 that occurs in fibroblasts as well as in the BB of an intestinal Na(+)-absorptive cell.

Nucleotides and Phosphorylation Bi-directionally Modulate Ca2+/Calmodulin-dependent Protein Kinase II (CaMKII) Binding to the N-Methyl-d-aspartate (NMDA) Receptor Subunit GluN2B

The Ca(2+)/calmodulin (CaM)-dependent protein kinase II (CaMKII) and the NMDA-type glutamate receptor are key regulators of synaptic plasticity underlying learning and memory. Direct binding of CaMKII to the NMDA receptor subunit GluN2B (formerly known as NR2B) (i) is induced by Ca(2+)/CaM but outlasts this initial Ca(2+)-stimulus, (ii) mediates CaMKII translocation to synapses, and (iii) regulates synaptic strength. CaMKII binds to GluN2B around S1303, the major CaMKII phosphorylation site on GluN2B. We show here that a phospho-mimetic S1303D mutation inhibited CaM-induced CaMKII binding to GluN2B in vitro, presenting a conundrum how binding can occur within cells, where high ATP concentration should promote S1303 phosphorylation. Surprisingly, addition of ATP actually enhanced the binding. Mutational analysis revealed that this positive net effect was caused by four modulatory effects of ATP, two positive (direct nucleotide binding and CaMKII T286 autophosphorylation) and two negative (GluN2B S1303 phosphorylation and CaMKII T305/6 autophosphorylation). Imaging showed positive regulation by nucleotide binding also within transfected HEK cells and neurons. In fact, nucleotide binding was a requirement for efficient CaMKII interaction with GluN2B in cells, while T286 autophosphorylation was not. Kinetic considerations support a model in which positive regulation by nucleotide binding and T286 autophosphorylation occurs faster than negative modulation by GluN2B S1303 and CaMKII T305/6 phosphorylation, allowing efficient CaMKII binding to GluN2B despite the inhibitory effects of the two slower reactions.

Role of the CaMKII/NMDA Receptor Complex in the Maintenance of Synaptic Strength

During long-term potentiation (LTP), synapses undergo stable changes in synaptic strength. The molecular memory processes that maintain strength have not been identified. One hypothesis is that the complex formed by the Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and the NMDA-type glutamate receptor (NMDAR) is a molecular memory at the synapse. To establish a molecule as a molecular memory, it must be shown that interfering with the molecule produces a persistent reversal of LTP. We used the CN class of peptides that inhibit CaMKII binding to the NR2B subunit in vitro to test this prediction in rat hippocampal slices. We found that CN peptides can reverse saturated LTP, allowing additional LTP to be induced. The peptide also produced a persistent reduction in basal transmission. We then tested whether CN compounds actually affect CaMKII binding in living cells. Application of CN peptide to slice cultures reduced the amount of CaMKII concentrated in spines, consistent with delocalization of the kinase from a binding partner in the spine. To more specifically assay the binding of CaMKII to the NMDAR, we used coimmunoprecipitation methods. We found that CN peptide decreased synaptic strength only at concentrations necessary to disrupt the CaMKII/NMDAR complex, but not at lower concentrations sufficient to inhibit CaMKII activity. Importantly, both the reduction of the complex and the reduction of synaptic strength persisted after removal of the inhibitor. These results support the hypothesis that the CaMKII/NMDAR complex has switch-like properties that are important in the maintenance of synaptic strength.