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1.
Widely regarded as a specific and potent inhibitor of CaM kinases, especially CaMKII, KN93 has long been used to investigate the possible roles of CaMKII in a wide range of biological functions and systems, such as cultured cells, primary neurons, and brain slices. However, here we present evidence showing that KN93 and its structural analog KN92, which does not inhibit CaMKII, exert an unexpected, reversible, and specific reduction of currents of L-type calcium channels (CaV1.3 and CaV1.2), as compared to N-type calcium channels (CaV2.2). This effect is dependent not only on incubation time, but also on the dose of KN93 or KN92. Moreover, the effect appears to be independent of endocytosis, exocytosis, and proteasome activity. Washout and return to normal media rescues the L channel currents. Conversely, the structurally unrelated CaMKII inhibitor, AIP, fails to mimic the KN93/KN92 effect on L channel currents. Together, our data suggest that, in addition to inhibiting CaMKII, KN93 also affects CaV1.3 and CaV1.2 calcium channels in a CaMKII-independent manner. 相似文献
2.
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 V2.1 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 V2.1 channel and synapsin-1 simultaneously. CaMKII binding to Ca V2.1 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 V2.1 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 V2.1 channels. These results define the functional properties of a signaling complex of CaMKII and Ca V2.1 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. 相似文献
4.
Calcium/calmodulin-dependent protein kinase II (CaMKII) is a multifunctional serine/threonine protein kinase that transmits calcium signals in various cellular processes. CaMKII is activated by calcium-bound calmodulin (Ca 2+/CaM) through a direct binding mechanism involving a regulatory C-terminal α-helix in CaMKII. The Ca 2+/CaM binding triggers transphosphorylation of critical threonine residues proximal to the CaM-binding site leading to the autoactivated state of CaMKII. The demonstration of its critical roles in pathophysiological processes has elevated CaMKII to a key target in the management of numerous diseases. The molecule KN-93 is the most widely used inhibitor for studying the cellular and in vivo functions of CaMKII. It is widely believed that KN-93 binds directly to CaMKII, thus preventing kinase activation by competing with Ca 2+/CaM. Herein, we employed surface plasmon resonance, NMR, and isothermal titration calorimetry to characterize this presumed interaction. Our results revealed that KN-93 binds directly to Ca 2+/CaM and not to CaMKII. This binding would disrupt the ability of Ca 2+/CaM to interact with CaMKII, effectively inhibiting CaMKII activation. Our findings also indicated that KN-93 can specifically compete with a CaMKIIδ-derived peptide for binding to Ca 2+/CaM. As indicated by the surface plasmon resonance and isothermal titration calorimetry data, apparently at least two KN-93 molecules can bind to Ca 2+/CaM. Our findings provide new insight into how in vitro and in vivo data obtained with KN-93 should be interpreted. They further suggest that other Ca 2+/CaM-dependent, non-CaMKII activities should be considered in KN-93–based mechanism-of-action studies and drug discovery efforts. 相似文献
5.
The calcium channel blocker (CCB), nifedipine, is a more effective treatment for early‐ than late‐stage cardiac hypertrophy. We investigated the effects of early‐ and late‐stage nifedipine administration on calcium homeostasis, CaMKII (Ca 2+/calmodulin‐dependent protein kinase II) activity and apoptosis of cardiomyocytes under hypertrophic stimulation with angiotensin II (AngII). Primary rat cardiomyocytes were divided into five treatment groups: AK, AngII plus the CaMKII inhibitor, KN‐93; AN‐1 (early‐stage), AngII plus nifedipine × 48 h; AN‐2 (late‐stage), AngII × 48 h, then AngII plus nifedipine × 48 h; C, untreated; and A, AngII × 48 h. The t1/2β [time required for intracellular Ca 2+ concentration ([Ca 2+]i) to decline to one half of the peak value] decreased; however, CaMKII and SERCA2a (sarcoplasmic reticulum Ca 2+‐ATPase 2a) activities increased in the AN‐1 group compared with the AK group. In the AN‐2 group compared with the AN‐1 group, CaMKII activity, t1/2α [time required for [Ca 2+]i to increase from the bottom to one half of peak value], t1/2β, and apoptosis increased. These results indicate that the timing of CCB administration affects the calcium concentration and apoptosis of hypertrophic cardiomyocytes through the CaMKII–SERCA2a signalling pathway, thereby influencing the drug's protective activity against cardiomyocyte hypertrophy. Copyright © 2016 John Wiley & Sons, Ltd. 相似文献
7.
Cardiovascular complications are leading causes of morbidity and mortality in patients with chronic kidney disease (CKD). CKD significantly affects cardiac calcium (Ca 2+) regulation, but the underlying mechanisms are not clear. The present study investigated the modulation of Ca 2+ homeostasis in CKD mice. Echocardiography revealed impaired fractional shortening (FS) and stroke volume (SV) in CKD mice. Electrocardiography showed that CKD mice exhibited longer QT interval, corrected QT (QTc) prolongation, faster spontaneous activities, shorter action potential duration (APD) and increased ventricle arrhythmogenesis, and ranolazine (10 µmol/L) blocked these effects. Conventional microelectrodes and the Fluo-3 fluorometric ratio techniques indicated that CKD ventricular cardiomyocytes exhibited higher Ca 2+ decay time, Ca 2+ sparks, and Ca 2+ leakage but lower [Ca 2+] i transients and sarcoplasmic reticulum Ca 2+ contents. The CaMKII inhibitor KN93 and ranolazine (RAN; late sodium current inhibitor) reversed the deterioration in Ca 2+ handling. Western blots revealed that CKD ventricles exhibited higher phosphorylated RyR2 and CaMKII and reduced phosphorylated SERCA2 and SERCA2 and the ratio of PLB-Thr17 to PLB. In conclusions, the modulation of CaMKII, PLB and late Na + current in CKD significantly altered cardiac Ca 2+ regulation and electrophysiological characteristics. These findings may apply on future clinical therapies. 相似文献
8.
Cellular oxidative stress, associated with a variety of common cardiac diseases, is well recognized to affect the function of several key proteins involved in Ca 2+ signaling and excitation-contraction coupling, which are known to be exquisitely sensitive to reactive oxygen species. These include the Ca 2+ release channels of the sarcoplasmic reticulum (ryanodine receptors or RyR2s) and the Ca 2+/calmodulin-dependent protein kinase II (CaMKII). Oxidation of RyR2s was found to increase the open probability of the channel, whereas CaMKII can be activated independent of Ca 2+ through oxidation. Here, we investigated how oxidative stress affects RyR2 function and SR Ca 2+ signaling in situ, by analyzing Ca 2+ sparks in permeabilized mouse cardiomyocytes under a broad range of oxidative conditions. The results show that with increasing oxidative stress Ca 2+ spark duration is prolonged. In addition, long and very long-lasting (up to hundreds of milliseconds) localized Ca 2+ release events started to appear, eventually leading to sarcoplasmic reticulum (SR) Ca 2+ depletion. These changes of release duration could be prevented by the CaMKII inhibitor KN93 and did not occur in mice lacking the CaMKII-specific S2814 phosphorylation site on RyR2. The appearance of long-lasting Ca 2+ release events was paralleled by an increase of RyR2 oxidation, but also by RyR-S2814 phosphorylation, and by CaMKII oxidation. Our results suggest that in a strongly oxidative environment oxidation-dependent activation of CaMKII leads to RyR2 phosphorylation and thereby contributes to the massive prolongation of SR Ca 2+ release events. 相似文献
9.
Calmodulin (CaM) is a ubiquitous second messenger protein that regulates a variety of structurally and functionally diverse targets in response to changes in Ca 2+ concentration. CaM-dependent protein kinase II (CaMKII) and calcineurin (CaN) are the prominent CaM targets that play an opposing role in many cellular functions including synaptic regulation. Since CaMKII and CaN compete for the available Ca 2+/CaM, the differential affinity of these enzymes for CaM is crucial for achieving a balance in Ca 2+ signaling. We used the computational protein design approach to modify CaM binding specificity for these two targets. Starting from the X-ray structure of CaM in complex with the CaM-binding domain of CaMKII, we optimized CaM interactions with CaMKII by introducing mutations into the CaM sequence. CaM optimization was performed with a protein design program, ORBIT, using a modified energy function that emphasized intermolecular interactions in the sequence selection procedure. Several CaM variants were experimentally constructed and tested for binding to the CaMKII and CaN peptides using the surface plasmon resonance technique. Most of our CaM mutants demonstrated small increase in affinity for the CaMKII peptide and substantial decrease in affinity for the CaN peptide compared to that of wild-type CaM. Our best CaM design exhibited an about 900-fold increase in binding specificity towards the CaMKII peptide, becoming the highest specificity switch achieved in any protein-protein interface through the computational protein design approach. Our results show that computational redesign of protein-protein interfaces becomes a reliable method for altering protein binding affinity and specificity. 相似文献
10.
In cardiac and skeletal myocytes, and in most neurons, the opening of voltage‐gated Na + channels (Na V channels) triggers action potentials, a process that is regulated via the interactions of the channels’ intercellular C‐termini with auxiliary proteins and/or Ca 2+. The molecular and structural details for how Ca 2+ and/or auxiliary proteins modulate Na V channel function, however, have eluded a concise mechanistic explanation and details have been shrouded for the last decade behind controversy about whether Ca 2+ acts directly upon the Na V channel or through interacting proteins, such as the Ca 2+ binding protein calmodulin (CaM). Here, we review recent advances in defining the structure of Na V intracellular C‐termini and associated proteins such as CaM or fibroblast growth factor homologous factors (FHFs) to reveal new insights into how Ca 2+ affects Na V function, and how altered Ca 2+‐dependent or FHF‐mediated regulation of Na V channels is perturbed in various disease states through mutations that disrupt CaM or FHF interaction. 相似文献
11.
In this review article we give an overview of current knowledge with respect to redox-sensitive alterations in Na + and Ca 2+ handling in the heart. In particular, we focus on redox-activated protein kinases including cAMP-dependent protein kinase A (PKA), protein kinase C (PKC), and Ca/calmodulin-dependent protein kinase II (CaMKII), as well as on redox-regulated downstream targets such as Na + and Ca 2+ transporters and channels. We highlight the pathological and physiological relevance of reactive oxygen species and some of its sources (such as NADPH oxidases, NOXes) for excitation—contraction coupling (ECC). A short outlook with respect to the clinical relevance of redox-dependent Na + and Ca 2+ imbalance will be given. 相似文献
12.
Sudden cardiac death due to ventricular arrhythmias is a major problem. Drug therapies to prevent SCD do not provide satisfying results, leading to the demand for new antiarrhythmic strategies. New targets include Ca 2+/Calmodulin-dependent protein kinase II (CaMKII), the Na/Ca exchanger (NCX), the Ryanodine receptor (RyR, and its associated protein FKBP12.6 (Calstabin)) and the late component of the sodium current ( I Na-Late ), all related to intracellular calcium (Ca 2+) handling. In this review, drugs interfering with these targets (SEA-0400, K201, KN-93, W7, ranolazine, sophocarpine, and GS-967) are evaluated and their future as clinical compounds is considered. These new targets prove to be interesting; however more insight into long-term drug effects is necessary before clinical applicability becomes reality. 相似文献
13.
Cardiomyocyte apoptosis contributes to the development of coxsackievirus B3 (CVB3)‐induced myocarditis, but the mechanism for the apoptosis by CVB3 infection remains unclear. Here, we showed that CVB3‐induced endoplasmic reticulum (ER) stress response and apoptosis in cultured H9c2 cardiomyocytes. We found that Ca 2+‐calmodulin‐dependent kinase II (CaMKII) was activated by ER stress‐dependent intracellular Ca 2+ overload in the CVB3‐infected H9c2 cardiomyocytes. Treatment with an inhibitor of ER stress, 4‐phenylbutyric acid (4‐PBA), attenuated intracellular Ca 2+ accumulation indirectly and reduced CaMKII activity. Inhibition of CaMKII with pharmacological inhibitor (KN‐93) or short hairpin RNA reduced CVB3‐induced H9c2 apoptosis and repressed cytochrome c release from mitochondria to cytoplasm; whereas overexpression of the activated mutant of CaMKII (CaMKII‐T287D) enhanced CVB3‐induced H9c2 apoptosis and mitochondrial cytochrome c release, which could be alleviated by blocking of mitochondrial Ca 2+ uniporter or mitochondrial permeability transition pore. Further in vivo investigation revealed that blocking of CaMKII with KN‐93 prevented cardiomyocytes apoptosis and improved cardiac contractile function in CVB3‐infected mouse heart. Collectively, these findings provide a novel evidence that CaMKII plays a vital role in the promotion of CVB3‐induced cardiomyocyte apoptosis, which links ER stress and mitochondrial Ca 2+ uptake. 相似文献
14.
A hallmark feature of Ca 2+/calmodulin (CaM)-dependent protein kinase II (CaMKII) regulation is the generation of Ca 2+-independent autonomous activity by Thr-286 autophosphorylation. CaMKII autonomy has been regarded a form of molecular memory and is indeed important in neuronal plasticity and learning/memory. Thr-286-phosphorylated CaMKII is thought to be essentially fully active (∼70–100%), implicating that it is no longer regulated and that its dramatically increased Ca 2+/CaM affinity is of minor functional importance. However, this study shows that autonomy greater than 15–25% was the exception, not the rule, and required a special mechanism (T-site binding; by the T-substrates AC2 or NR2B). Autonomous activity toward regular R-substrates (including tyrosine hydroxylase and GluR1) was significantly further stimulated by Ca 2+/CaM, both in vitro and within cells. Altered Km and Vmax made autonomy also substrate- (and ATP) concentration-dependent, but only over a narrow range, with remarkable stability at physiological concentrations. Such regulation still allows molecular memory of previous Ca 2+ signals, but prevents complete uncoupling from subsequent cellular stimulation. 相似文献
15.
NO is known to modulate calcium handling and cellular signaling in the myocardium, but key targets for NO in the heart remain unidentified. Recent reports have implied that NO can activate calcium/calmodulin (Ca 2+/CaM)-dependent protein kinase II (CaMKII) in neurons and the heart. Here we use our novel sensor of CaMKII activation, Camui, to monitor changes in the conformation and activation of cardiac CaMKII (CaMKIIδ) activity after treatment with the NO donor S-nitrosoglutathione (GSNO). We demonstrate that exposure to NO after Ca 2+/CaM binding to CaMKIIδ results in autonomous kinase activation, which is abolished by mutation of the Cys-290 site. However, exposure of CaMKIIδ to GSNO prior to Ca 2+/CaM exposure strongly suppresses kinase activation and conformational change by Ca 2+/CaM. This NO-induced inhibition was ablated by mutation of the Cys-273 site. We found parallel effects of GSNO on CaM/CaMKIIδ binding and CaMKIIδ-dependent ryanodine receptor activation in adult cardiac myocytes. We conclude that NO can play a dual role in regulating cardiac CaMKIIδ activity. 相似文献
16.
Voltage-gated sodium channels (VGSCs) are fundamental to the initiation and propagation of action potentials in excitable cells. Ca2+/calmodulin (CaM) binds to VGSC type II (NaV1.2) isoleucine and glutamine (IQ) motif. An autism-associated mutation in NaV1.2 IQ motif, Arg1902Cys (R1902C), has been reported to affect the combination between CaM and the IQ motif compared to that of the wild type IQ motif. However, the detailed properties for the Ca2+-regulated binding of CaM to NaV1.2 IQ (1901Lys-1927Lys, IQwt) and mutant IQ motif (IQR1902C) remains unclear. Here, the binding ability of CaM and CaM's constituent proteins including N- and C lobe to the IQ motif of NaV1.2 and its mutant was investigated by protein pull-down experiments. We discovered that the combination between CaM and the IQ motif was U-shaped with the highest at [Ca2+] ≈ free and the lowest at 100 nM [Ca2+]. In the IQR1902C mutant, Ca2+-dependence of CaM binding was nearly lost. Consequently, the binding of CaM to IQR1902C at 100 and 500 nM [Ca2+] was increased compared to that of IQwt. Both N- and C lobe of CaM could bind with NaV1.2 IQ motif and IQR1902C mutant, with the major effect of C lobe. Furthermore, CaMKII had no impact on the binding between CaM and NaV1.2 IQ motif. This research offers novel insight to the regulation of NaV1.2 IQwt and IQR1902C motif, an autism-associated mutation, by CaM. 相似文献
17.
In cardiac muscle, the release of calcium ions from the sarcoplasmic reticulum through ryanodine receptor ion channels (RyR2s) leads to muscle contraction. RyR2 is negatively regulated by calmodulin (CaM) and by phosphorylation of Ca 2+/CaM-dependent protein kinase II (CaMKII). Substitution of three amino acid residues in the CaM binding domain of RyR2 (RyR2-W3587A/L3591D/F3603A, RyR2 ADA) impairs inhibition of RyR2 by CaM and results in cardiac hypertrophy and early death of mice carrying the RyR2 ADA mutation. To test the cellular function of CaMKII in cardiac hypertrophy, mutant mice were crossed with mice expressing the CaMKII inhibitory AC3-I peptide or the control AC3-C peptide in the myocardium. Inhibition of CaMKII by AC3-I modestly reduced CaMKII-dependent phosphorylation of RyR2 at Ser-2815 and markedly reduced CaMKII-dependent phosphorylation of SERCA2a regulatory subunit phospholamban at Thr-17. However the average life span and heart-to-body weight ratio of Ryr2ADA/ADA mice expressing the inhibitory peptide were not altered compared to control mice. In Ryr2ADA/ADA homozygous mice, AC3-I did not alter cardiac morphology, enhance cardiac function, improve sarcoplasmic reticulum Ca 2+ handling, or suppress the expression of genes implicated in cardiac remodeling. The results suggest that CaMKII was not required for the rapid development of cardiac hypertrophy in Ryr2ADA/ADA mice. 相似文献
18.
We have shown earlier a requirement for Ca 2+ and calmodulin (CaM) in the H 2O 2-induced activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and protein kinase B (PKB), key mediators of growth-promoting, proliferative, and hypertrophic responses in vascular smooth muscle cells (VSMC). Because the effect of CaM is mediated through CaM-dependent protein kinase II (CaMKII), we have investigated here the potential role of CaMKII in H 2O 2-induced ERK1/2 and PKB phosphorylation by using pharmacological inhibitors of CaM and CaMKII, a CaMKII inhibitor peptide, and siRNA knockdown strategies for CaMKIIα. Calmidazolium and W-7, antagonists of CaM, as well as KN-93, a specific inhibitor of CaMKII, attenuated H 2O 2-induced responses of ERK1/2 and PKB phosphorylation in a dose-dependent fashion. Similar to H 2O 2, calmidazolium and KN-93 also exhibited an inhibitory effect on glucose/glucose oxidase-induced phosphorylation of ERK1/2 and PKB in these cells. Transfection of VSMC with CaMKII autoinhibitory peptide corresponding to the autoinhibitory domain (aa 281–309) of CaMKII and with siRNA of CaMKIIα attenuated the H 2O 2-induced phosphorylation of ERK1/2 and PKB. In addition, calmidazolium and KN-93 blocked H 2O 2-induced Pyk2 and insulin-like growth factor-1 receptor (IGF-1R) phosphorylation. Moreover, treatment of VSMC with CaMKIIα siRNA abolished the H 2O 2-induced IGF-1R phosphorylation. H 2O 2 treatment also induced Thr 286 phosphorylation of CaMKII, which was inhibited by both calmidazolium and KN-93. These results demonstrate that CaMKII plays a critical upstream role in mediating the effects of H 2O 2 on ERK1/2, PKB, and IGF-1R phosphorylation. 相似文献
19.
Altered insulin secretion contributes to the pathogenesis of type 2 diabetes. This alteration is correlated with altered intracellular Ca 2+-handling in pancreatic β cells. Insulin secretion is triggered by elevation in cytoplasmic Ca 2+ concentration ([Ca 2+] cyt) of β cells. This elevation in [Ca 2+] cyt leads to activation of Ca 2+/calmodulin-dependent protein kinase II (CAMKII), which, in turn, controls multiple aspects of insulin secretion. CaMKII is known to phosphorylate ryanodine receptor 2 (RyR2), an intracellular Ca 2+-release channel implicated in Ca 2+-dependent steps of insulin secretion. Our data show that RyR2 is CaMKII phosphorylated in a pancreatic β-cell line in a glucose-sensitive manner. However, it is not clear whether any change in CaMKII-mediated phosphorylation underlies abnormal RyR2 function in β cells and whether such a change contributes to alterations in insulin secretion. Therefore, knock-in mice with a mutation in RyR2 that mimics its constitutive CaMKII phosphorylation, RyR2-S2814D, were studied. This mutation led to a gain-of-function defect in RyR2 indicated by increased basal RyR2-mediated Ca 2+ leak in islets of these mice. This chronic in vivo defect in RyR2 resulted in basal hyperinsulinemia. In addition, S2814D mice also developed glucose intolerance, impaired glucose-stimulated insulin secretion and lowered [Ca 2+] cyt transients, which are hallmarks of pre-diabetes. The glucose-sensitive Ca 2+ pool in islets from S2814D mice was also reduced. These observations were supported by immunohistochemical analyses of islets in diabetic human and mouse pancreata that revealed significantly enhanced CaMKII phosphorylation of RyR2 in type 2 diabetes. Together, these studies implicate that the chronic gain-of-function defect in RyR2 due to CaMKII hyperphosphorylation is a novel mechanism that contributes to pathogenesis of type 2 diabetes. 相似文献
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