Overexpression Of Calmodulin Research Articles

Investigating the Impact of Electrostatic Interactions on Calmodulin Binding and Ca2+-Dependent Activation of the Calcium-Gated Potassium SK4 Channel.

Ca2+ binding to the ubiquitous Ca2+ sensing protein calmodulin (CaM) activates the intermediate conductance Ca2+-activated SK4 channel. Potential hydrophilic pockets for CaM binding have been identified at the intracellular HA and HB helices in the C-terminal of SK4 from the three published cryo-EM structures of SK4. Single charge reversal substitutions at either site, significantly weakened the pull-down of SK4 by CaM wild-type (CaM), and decreased the TRAM-34 sensitive outward K+ current densities in native HEK293T cells when compared with SK4 WT measured under the same conditions. Only the doubly substituted SK4 R352D/R355D (HB helix) obliterated the CaM-mediated pull-down and thwarted outward K+ currents. However, overexpression of CaM E84K/E87K, which had been predicted to face the arginine doublet, restored the CaM-mediated pull-down of SK4 R352D/R355D and normalized its whole-cell current density. Virtual analysis of the putative salt bridges supports a unique role for the positively charged arginine doublet at the HB helix into anchoring the interaction with the negatively charged CaM glutamate 84 and 87 CaM. Our findings underscore the unique contribution of electrostatic interactions in carrying CaM binding onto SK4 and support the role of the C-terminal HB helix to the Ca2+-dependent gating process.

A CACNA1C variant associated with cardiac arrhythmias provides mechanistic insights in the calmodulation of L-type Ca2+ channels

We recently reported the identification of a de novo single nucleotide variant in exon 9 of CACNA1C associated with prolonged repolarization interval. Recombinant expression of the glycine to arginine variant at position 419 produced a gain in the function of the L-type CaV1.2 channel with increased peak current density and activation gating but without significant decrease in the inactivation kinetics. We herein reveal that these properties are replicated by overexpressing calmodulin (CaM) with CaV1.2 WT and are reversed by exposure to the CaM antagonist W-13. Phosphomimetic (T79D or S81D), but not phosphoresistant (T79A or S81A), CaM surrogates reproduced the impact of CaM WT on the function of CaV1.2 WT. The increased channel activity of CaV1.2 WT following overexpression of CaM was found to arise in part from enhanced cell surface expression. In contrast, the properties of the variant remained unaffected by any of these treatments. CaV1.2 substituted with the α-helix breaking proline residue were more reluctant to open than CaV1.2 WT but were upregulated by phosphomimetic CaM surrogates. Our results indicate that (1) CaM and its phosphomimetic analogs promote a gain in the function of CaV1.2 and (2) the structural properties of the first intracellular linker of CaV1.2 contribute to its CaM-induced modulation. We conclude that the CACNA1C clinical variant mimics the increased activity associated with the upregulation of CaV1.2 by Ca2+-CaM, thus maintaining a majority of channels in a constitutively active mode that could ultimately promote ventricular arrhythmias.

Abstract 890: Calmodulin governs nuclear entry of fusion PAX3/FOXO1 oncoprotein, a target in alveolar rhabdomyosarcoma

Abstract Reciprocal chromosomal translocations resulting in the fusion of the DNA binding domain of the transcription factor PAX3 and the transactivational domain of FOXO1 underlie most cases of alveolar rhabdomyosarcoma (aRMS). Testing of compound MTPF63 against a panel of sarcoma cell lines showed that it had an inhibitory effect on proliferation of PAX3-FOXO1 translocation-positive cells. Further evaluation of its activity in Rh4 cells transduced with a PAX3-FOXO1 luciferase reporter showed that it attenuated reporter activity. Additional confirmation for the compound's inhibitory impact on PAX3-FOXO1 activity was obtained by detecting significant attenuation in the levels of Alk1 and MyoD1 proteins whose expression is driven by PAX3-FOXO1 in aRMS cells. Cell viability assays performed under 2D and 3D culturing conditions demonstrated that MTPF63 inhibits cell growth more effectively in translocation-positive alveolar RMS than in translocation-negative embryonal RMS. A hypothesis for MTPF63's mode of action was generated using the complete pool of barcoded essential heterozygous (~1150 strains) and homozygous (~4800 strains) diploid gene deletion mutants of Saccharomyces cerevisiae to identify gene deletions that confer sensitivity to the compound. Gene ontology enrichment analysis of 255 sensitive mutants whose growth rates were significantly inhibited in the presence of the compound compared to control, not only revealed calmodulin as main target of the compound but also illustrated the compound's interference with calmodulin-dependent nuclear protein import pathway, suggesting that it might impact PAX3-FOXO1 nuclear localization. Immunostaining of nuclear-localized PAX3-FOXO1 oncoprotein in two aRMS cell lines revealed cytoplasmic localization. Immunoblot analysis of fractionated cytosolic and nuclear protein lysates for PAX3-FOXO1 in MTPF63 treated cells also showed increased amounts in the cytosolic fractions. Transient overexpression of calmodulin was shown to partially rescue the inhibitory effect of the compound on PAX3-FOXO1-driven luciferase expression in RH4 reporter cells. Taken together, the data demonstrate the involvement of calmodulin in the nuclear import of PAX3-FOXO1 and introduce calmodulin antagonists as potential therapeutics against translocation-positive childhood alveolar rhabdomyosarcoma. Citation Format: Emad Darvishi, Cheryl Lynn Thomas, Berkley Eric Gryder, John Frederick Shern, Javed Khan, Girma M Woldemichael. Calmodulin governs nuclear entry of fusion PAX3/FOXO1 oncoprotein, a target in alveolar rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 890.

UVB induces apoptosis via downregulation of CALML3-dependent JNK1/2 and ERK1/2 pathways in cataract.

The aim of the current study was to understand the mechanisms of apoptosis occurring in cultured human lens epithelial cells(HLECs) following ultravioletB (UVB) irradiation. The investigations intended to confirm the presence of apoptosis and to reveal the roles of oxidative stress, calcium (Ca2+), c‑Jun NH2‑terminal kinase (JNK)1/2, and extracellular signal‑regulated kinase (ERK)1/2 signaling pathway in these progresses. Cell apoptosis, ROS generation and intracellular Ca2+ concentration was measured by flow cytometry. The expression of CALML3, caspase-3, Bax, Bcl-2, p-JNK1/2, JNK1/2, p-ERK1/2 and ERK1/2 was measured by RT-qPCR and western blot analysis. AnnexinV‑fluorescein isothiocyanate/propidium iodide staining demonstrated that UVB irradiation increased the apoptotic rate, reactive oxygen species(ROS) production and intracellular Ca2+ concentration of HLECs in dose‑ and time‑dependent manners. Overexpression of calmodulin like3 (CALML3) reversed the effects of UVB irradiation on apoptosis, ROS production and Ca2+ concentration of HLECs, and decreased expressions of caspase‑3 and Bax, with increased expressions of Bcl‑2. Notably, silencing of CALML3 had similar effects to UVB irradiation and inhibited the activation JNK1/2 and ERK1/2 pathways. Nimodipine, a Ca2+‑channel antagonist, significantly attenuated the damages induced by CALML3 downregulation. In conclusion, UVB irradiation induced increase in apoptosis, ROS production and Ca2+ concentration of HLECs, in part, by downregulating the expression of CALML3 and involved oxidative stress, Ca2+, JNK1/2 and ERK1/2 signaling pathways, suggesting that investigating CALML3 may useful for developing cataract treatment.

Effect of aluminum stress on the expression of calmodulin and the role of calmodulin in aluminum tolerance

Calmodulin (CaM) is a calcium ion-binding protein that regulates a variety of cellular functions through its downstream target proteins. Previous studies have reported that overexpression of CaM enhances tolerance to stress, including resistance to salt, heat, cold, drought and plant pathogens. In this study, the growth of Cryptococcus humicola was inhibited by the CaM inhibitor, trifluoperazine, under aluminum (Al) stress. The expression of CaM of C. humicola (ChCaM) was upregulated when the concentration and treatment time with Al was increased. These results indicate that Al stress affects the transcription and translation of ChCaM and that ChCaM may play an important role in Al tolerance. Transgenic ChCaM Saccharomyces cerevisiae was constructed and designated as Sc-ChCaM. The ability of Sc-ChCaM to develop resistance to Al was significantly higher than that of control yeast containing the empty vector pYES3/CT designated as Sc. The residual Al content in the medium containing ChCaM transgenic yeast in culture was significantly lower than the initial amount of Al added in the medium or the residual Al content in the medium containing the control yeast in culture. This finding suggests that ChCaM confers Al tolerance in transgenic yeast, and the absorption of active Al from the culture may be one reason for Al tolerance. These results indicate that ChCaM may be a candidate gene for Al tolerance in engineered plants.

Effects of calmodulin on expression of lignin-modifying enzymes in Pleurotus ostreatus.

Previously, we suppressed the expression of genes encoding isozymes of lignin peroxidase (LiP) and manganese peroxidase (MnP) using a calmodulin (CaM) inhibitor, W7, in the white-rot fungus Phanerochaete chrysosporium; this suggested that CaM positively regulates their expression. Here, we studied the role of CaM in another white-rot fungus, Pleurotus ostreatus, which produces MnP and versatile peroxidase (VP), but not LiP. W7 upregulated Mn(2+)-dependent oxidation of guaiacol, suggesting that CaM negatively regulates the production of the enzymes. Suppression of CaM in P. ostreatus using RNAi also led to upregulation of enzyme activity, whereas overexpression of CaM in P. ostreatus caused downregulation. Real-time RT-PCR showed that MnP1-6 and VP3 levels in the CaM-knockdown strain were higher than those in the wild-type strain, while MnP-5 and -6 and VP1 and 2 levels in the CaM-overexpressing strain were lower than in the wild type. Moreover, we also found that another ligninolytic enzyme, laccase, which is not produced by P. chrysosporium, was negatively regulated by CaM in P. ostreatus similar to MnP and VP. Although overexpression of CaM did not reduce the ability of P. ostreatus to digest beech wood powder, the percentage of lignin remaining in the digest was slightly higher than in the wild-type strain digest.

Competitive and Non-competitive Regulation of Calcium-dependent Inactivation in CaV1.2 L-type Ca2+ Channels by Calmodulin and Ca2+-binding Protein 1

CaV1.2 interacts with the Ca(2+) sensor proteins, calmodulin (CaM) and calcium-binding protein 1 (CaBP1), via multiple, partially overlapping sites in the main subunit of CaV1.2, α1C. Ca(2+)/CaM mediates a negative feedback regulation of Cav1.2 by incoming Ca(2+) ions (Ca(2+)-dependent inactivation (CDI)). CaBP1 eliminates this action of CaM through a poorly understood mechanism. We examined the hypothesis that CaBP1 acts by competing with CaM for common interaction sites in the α1C- subunit using Förster resonance energy transfer (FRET) and recording of Cav1.2 currents in Xenopus oocytes. FRET detected interactions between fluorescently labeled CaM or CaBP1 with the membrane-attached proximal C terminus (pCT) and the N terminus (NT) of α1C. However, mutual overexpression of CaM and CaBP1 proved inadequate to quantitatively assess competition between these proteins for α1C. Therefore, we utilized titrated injection of purified CaM and CaBP1 to analyze their mutual effects. CaM reduced FRET between CaBP1 and pCT, but not NT, suggesting competition between CaBP1 and CaM for pCT only. Titrated injection of CaBP1 and CaM altered the kinetics of CDI, allowing analysis of their opposite regulation of CaV1.2. The CaBP1-induced slowing of CDI was largely eliminated by CaM, corroborating a competition mechanism, but 15-20% of the effect of CaBP1 was CaM-resistant. Both components of CaBP1 action were present in a truncated α1C where N-terminal CaM- and CaBP1-binding sites have been deleted, suggesting that the NT is not essential for the functional effects of CaBP1. We propose that CaBP1 acts via interaction(s) with the pCT and possibly additional sites in α1C.

The over-expression of calmodulin from Antarctic notothenioid fish increases cold tolerance in tobacco

Genes involved in the calcium signalling pathway have a relationship with cold tolerance in many plants. The primary reaction to many different environmental stresses is an increase in the cytoplasmic Ca2+ concentration. Such variations in the Ca2+ concentration could change the activity of Ca2+-dependent protein functions, further regulating the expression of stress-related genes; therefore, the Ca2+ signalling pathway is involved in the biological stress reaction. The expression of the calcium-modulated protein gene, calmodulin, in Antarctic notothenioid fish (Dissostichus mawsoni) accounts for 0.23% of all transcripts, which is a very high level of expression in this cold-water fish. To elucidate the function of calmodulin (CaM) from Antarctic notothenioid fishes, we introduced the calmodulin (CaM) gene into tobacco plants using a viral vector based on pea early browning virus (PEBV). RT-PCR and Western blot results confirmed that the CaM gene was over-expressed in tobacco. Under low-temperature stress, the CaM transgenic plants exhibited faster growth than wild-type plants. The physiological and biochemical effects of the high-level expression of CaM in tobacco were analysed, and the changes in the electrolyte leakage activity and malondialdehyde content showed that CaM over-expression in tobacco increased the cold tolerance of the plants. These results demonstrate that CaM can possibly be used to enhance the low-temperature tolerance of plants.

Interaction with calmodulin is important for the secretion of thimet oligopeptidase following stimulation

Thimet oligopeptidase (EC 3.4.24.15; EP24.15) was originally described as a neuropeptide-metabolizing enzyme, highly expressed in the brain, kidneys and neuroendocrine tissue. EP24.15 lacks a typical signal peptide sequence for entry into the secretory pathway and is secreted by cells via an unconventional and unknown mechanism. In this study, we identified a novel calcium-dependent interaction between EP24.15 and calmodulin, which is important for the stimulated, but not constitutive, secretion of EP24.15. We demonstrated that, in vitro, EP24.15 and calmodulin physically interact only in the presence of Ca2+, with an estimated Kd value of 0.52 mum. Confocal microscopy confirmed that EP24.15 colocalizes with calmodulin in the cytosol of resting HEK293 cells. This colocalization markedly increases when cells are treated with either the calcium ionophore A23187 or the protein kinase A activator forskolin. Overexpression of calmodulin in HEK293 cells is sufficient to greatly increase the A23187-stimulated secretion of EP24.15, which can be inhibited by the calmodulin inhibitor calmidazolium. The specific inhibition of protein kinase A with KT5720 reduces the A23187-stimulated secretion of EP24.15 and inhibits the synergistic effects of forskolin with A23187. Treatment with calmidazolium and KT5720 nearly abolishes the stimulatory effects of A23187 on EP24.15 secretion. Together, these data suggest that the interaction between EP24.15 and calmodulin is regulated within cells and is important for the stimulated secretion of EP24.15 from HEK293 cells.

Overexpression of Calmodulin in Pancreatic β Cells Induces Diabetic Nephropathy

Recently, endothelial dysfunction induced by an uncoupling of vascular endothelial growth factor (VEGF) and nitric oxide has been implicated in the pathogenesis of diabetic nephropathy (DN). Investigating the pathogenesis of DN has been limited, however, because of the lack of animal models that mimic the human disease. In this report, pancreatic beta cell-specific calmodulin-overexpressing transgenic (CaMTg) mice, a potential new model of DN, are characterized with particular emphasis on VEGF and related molecules. CaMTg mice developed hyperglycemia at 3 wk and persistent proteinuria by 3 mo. Morphometric analysis showed considerable increases in the glomerular and mesangial areas with deposition of type IV collagen. Moreover, the pathologic hallmarks of human DN (mesangiolysis, Kimmelstiel-Wilson-like nodular lesions, exudative lesions, and hyalinosis of afferent and efferent arteries with neovascularization) were observed. In addition, increased VEGF expression was associated with an increased number of peritubular capillaries. Expression of endothelial nitric oxidase synthase was reduced and that of VEGF was markedly elevated in CaMTg mice kidney compared with nontransgenic mice. No differences in VEGF receptor-1 or VEGF receptor-2 expression were observed between CaMTg mice and nontransgenic kidneys. In summary, CaMTg mice develop most of the distinguishing lesions of human DN, and the elevated VEGF expression in the setting of diminished endothelial nitric oxide synthase expression may lead to endothelial proliferation and dysfunction. This model may prove useful in the study of the pathogenesis and treatment of DN.

The Role of Calmodulin Recruitment in Ca2+ Stimulation of Adenylyl Cyclase Type 8

Ca2+ stimulation of adenylyl cyclase type 8 (AC8) is mediated by calmodulin (CaM). An earlier study identified two CaM binding sites in AC8; one that was apparently not essential for AC8 activity, located at the N terminus, and a second site that was critical for Ca2+ stimulation, found at the C terminus (Gu, C., and Cooper, D. M. F. (1999) J. Biol. Chem. 274, 8012-8021). This study explores the role of these two CaM binding domains and their interaction in regulating AC8 activity, employing binding and functional studies with mutant CaM and modified AC8 species. We report that the N-terminal CaM binding domain of AC8 has a role in recruiting CaM and that this recruitment is essential to permit stimulation by Ca2+ in vivo. Using Ca2+-insensitive mutants of CaM, we found that partially liganded CaM can bind to AC8, but only fully liganded Ca2+/CaM can stimulate AC8 activity. Moreover, partially liganded CaM inhibited AC8 activity in vivo. The results indicate that CaM pre-associates with the N terminus of AC8, and we suggest that this recruited CaM is used by the C terminus of AC8 to mediate Ca2+ stimulation.

Cyclosporin Inhibits Cardiomegaly in the Conditional, Cardiac GCaMP2 Mice

Mice expressing the Ca2+ sensor protein GCaMP2 in cardiac myocytes under the control of a doxocycline -regulated αMHC promoter (ccGC2 mice) develop cardiomegaly by 14 weeks. As the transgene consists of a calmodulin – eGFP chimeric protein, we performed initial experiments to examine the mechanism(s) underlying the development of cardiac hypertrophy. Previous observations suggest that the overexpression of calmodulin can lead to hypertrophic cardiomyopathy (Gruver et al Endocrin 1993), suggesting a possible activation of the calcineurin/nFAT pathway (Dong et al Cardio Res 2003, Molkentin et al Cell 1998). We examined whether inhibiting the activation of this pathway prevented GCaMP2 dependent development of cardiac hypertrophy. Beginning at post natal day 7, wildtype and ccGC2 litter mate controls were injected with either vehicle only or cyclosporin (10–20mg/kg) twice daily for two weeks. Mice were euthanized at 3 weeks of age, at which time heart and body weight were measured. The heart to body weight ratio in 3 week old ccGC2 mice injected with vehicle was 15% greater than vehicle injected wildtype mice or ccGC2 mice injected with cyclosporin A (5.8 ± 0.2 n=5; 5.1 ± 0.2 n=5; 5.2 ± 0.1 n=7 p < 0.02, respectively). Cardiomegaly was inhibited in ccGC2 mice injected with cyclosporin A when compared to cyclosporin A injected wildtype litter mate controls (5.2 ± 0.1 n=7; 5.3 ± 0.1 n=6, respectively). These data suggest that GCaMP2 transgene expression produces cardiac hypertrophy through stimulation of of calcineurin pathway. As the transgene is temporally regulated, ccGC2 mice may be useful in examining the signaling processes leading to the development of cardiomegaly in adult mice. Values are ± SE. NIH grants HL45239 and DK58795.

Overexpression of calmodulin induces cardiac hypertrophy by a calcineurin-dependent pathway

The possible role of calcineurin in cardiac hypertrophy induced by calmodulin (CaM) overexpression in the heart was investigated. CaM transgenic (CaM-TG) mice developed marked cardiac hypertrophy and exhibited up-regulation of atrial natriuretic factor (ANF) and β-myosin heavy chain gene expression in the heart during the first 2 weeks after birth. The activity of calcineurin in the heart was also significantly increased in CaM-TG mice compared with wild-type littermates. Treatment of CaM-TG mice with the calcineurin inhibitor FK506 (1 mg/kg per day) prevented the increase in the heart-to-body weight ratio as well as that in cardiomyocyte width. FK506 also inhibited the induction of fetal-type cardiac gene expression in CaM-TG mice. Overexpression of CaM in cultured rat cardiomyocytes activated the ANF gene promoter in a manner sensitive to FK506. Activation of a calcineurin-dependent pathway thus contributes to the development of cardiac hypertrophy induced by CaM overexpression in the heart.

Calmodulin physically interacts with the erythropoietin receptor and enhances Jak2-mediated signaling

Stimulation of the erythropoietin receptor (EpoR) induces a transient increase in intracellular Ca 2+ level as well as activation of the Jak2 tyrosine kinase to stimulate various downstream signaling pathways. Here, we demonstrate that the universal Ca 2+ receptor calmodulin (CaM) binds EpoR in a Ca 2+-dependent manner in vitro. Binding studies using various EpoR mutants in hematopoietic cells showed that CaM binds the membrane-proximal 65-amino-acid cytoplasmic region (amino acids 258–312) of EpoR that is critical for activation of Jak2-mediated EpoR signaling. Structurally unrelated CaM antagonists, W-13 and CMZ, inhibited activation of Jak2-mediated EpoR signaling pathways, whereas W-12, a W-13 analog, did not show any significant inhibitory effect. Moreover, overexpression of CaM augmented Epo-induced tyrosine phosphorylation of the EpoR. W-13, but not W-12, also inhibited Epo-induced proliferation and survival. Together, these results indicate that CaM binds to the membrane-proximal EpoR cytoplasmic region and plays an essential role in activation of Jak2-mediated EpoR signaling.

SK3-1C, a Dominant-negative Suppressor of SKCa and IKCa Channels

Small conductance Ca2+-activated K+ channels, products of the SK1-SK3 genes, regulate membrane excitability both within and outside the nervous system. We report the characterization of a SK3 variant (SK3-1C) that differs from SK3 by utilizing an alternative first exon (exon 1C) in place of exon 1A used by SK3, but is otherwise identical to SK3. Quantitative RT-PCR detected abundant expression of SK3-1C transcripts in human lymphoid tissues, skeletal muscle, trachea, and salivary gland but not the nervous system. SK3-1C did not produce functional channels when expressed alone in mammalian cells, but suppressed SK1, SK2, SK3, and IKCa1 channels, but not BKCa or KV channels. Confocal microscopy revealed that SK3-1C sequestered SK3 protein intracellularly. Dominant-inhibitory activity of SK3-1C was not due to a nonspecific calmodulin sponge effect since overexpression of calmodulin did not reverse SK3-1C-mediated intracellular trapping of SK3 protein, and calmodulin-Ca2+-dependent inactivation of CaV channels was not affected by SK3-1C overexpression. Deletion analysis identified a dominant-inhibitory segment in the SK3-1C C terminus that resembles tetramerization-coiled-coiled domains reported to enhance tetramer stability and selectivity of multimerization of many K+ channels. SK3-1C may therefore suppress calmodulin-gated SKCa/IKCa channels by trapping these channel proteins intracellularly via subunit interactions mediated by the dominant-inhibitory segment and thereby reduce functional channel expression on the cell surface. Such family-wide dominant-negative suppression by SK3-1C provides a powerful mechanism to titrate membrane excitability and is a useful approach to define the functional in vivo role of these channels in diverse tissues by their targeted silencing.

Calmodulin Binds to the C Terminus of Sodium Channels Nav1.4 and Nav1.6 and Differentially Modulates Their Functional Properties

Modulation of voltage-gated sodium channels (VGSC) can have a major impact on cell excitability. Analysis of calmodulin (CaM) binding to GST-fusion proteins containing the C-terminal domains of Nav1.1-Nav1.9 indicates that some of the tetrodotoxin-sensitive VGSC isoforms, including NaV1.4 and NaV1.6, are able to bind CaM in a calcium-independent manner. Here we demonstrate that association with CaM is important for functional expression of NaV1.4 and NaV1.6 VGSCs. Disrupting the interaction between CaM and the C terminus of NaV1.4 and NaV1.6 channels reduced current amplitude by 99 and 62%, respectively. Overexpression of CaM increased the current generated by Nav1.4 and Nav1.6 C-terminal mutant constructs that exhibited intermediate current densities and intermediate binding affinities for CaM, demonstrating that this effect on current density was directly dependent on the ability of the C terminus to bind CaM. In addition to the effects on current density, calmodulin also was able to modulate the inactivation kinetics of Nav1.6, but not Nav1.4, currents in a calcium-dependent manner. Our data demonstrate that CaM can regulate the properties of VGSCs via calcium-dependent and calcium-independent mechanisms and suggest that modulation of neuronal sodium channels may play a role in calcium-dependent neuronal plasticity.

Calmodulin regulation of excitation-contraction coupling in cardiac myocytes.

Calmodulin (CaM) as a ubiquitous Ca2+ sensor interacts with multiple key molecules involved in excitation-contraction (EC) coupling. In the present study, we report that adenoviral expression of a mutant CaM lacking all of its four Ca2+-binding sites, CaM(1-4), at a level 6.5-fold over endogenous CaM markedly increases the amplitude and abbreviates the decay time of Ca2+ transients and contraction in cultured rat ventricular myocytes. To determine the underlying mechanisms, we examined the properties of L-type Ca2+ channels, Ca2+/CaM-dependent protein kinase II (CaMKII), and phospholamban (PLB) in the sarcoplasmic reticulum (SR). We found that CaM(1-4) expression markedly augmented L-type Ca2+ current amplitude and slowed its inactivation. Surprisingly, overexpression of CaM(1-4) increased CaMKII activity and phosphorylation of PLB-Thr-17. Moreover, CaM(1-4) elevated diastolic Ca2+ and caffeine-labile Ca2+ content of the SR. Inhibition of CaMKII by KN-93 or a myristoylated autocamtide-2 related inhibitory peptide prevented the aforementioned PLB phosphorylation and reversed the positive inotropic and relaxant effects, indicating that CaMKII is essential to CaM(1-4) actions. These results demonstrate that CaM modulates Ca2+ influx, SR Ca2+ release, and Ca2+ recycling during cardiac EC coupling. A novel finding of this study is that expression of a Ca2+-insensitive CaM mutant can lead to activation of CaMKII in cardiac myocytes.

Calmodulin as a potential negative regulator of Arabidopsis COR gene expression.

Previous studies have implicated calmodulin as a signaling component required for the cold induction of COR ( c old o n r egulated) genes in Arabidopsis ([Braam and Davis, 1990][1]; [Tahtiharju et al., 1997][2]). Here, we present data that show that overexpression of calmodulin in planta causes

Binding of IRS proteins to calmodulin is enhanced in insulin resistance.

The IRS proteins, major endogenous targets of the insulin receptor, bind to calmodulin in a Ca(2+)-dependent manner. Here, we have examined the interaction between these proteins in animal and cultured cell models of insulin resistance. Both IRS-1 and IRS-2 co-immunoprecipitate with calmodulin from insulin target tissues in rats. The interaction between calmodulin and IRS proteins in rat soleus muscle was enhanced when insulin resistance was induced in rats by treatment with dexamethasone for 5 days. Moreover, injection of angiotensin II into the inferior vena cava enhanced the binding in rat cardiac muscle. Similarly, increased binding between calmodulin and IRS-1 was observed in isolated cells incubated with tumor necrosis factor-alpha. Overexpression of calmodulin in Chinese hamster ovary cells reduced the tyrosine phosphorylation of IRS-1 induced by insulin, with a concomitant decrease in insulin-stimulated association of IRS-1 with the 85-kDa regulatory subunit of phosphatidylinositol 3-kinase. Insulin-stimulated phosphatidylinositol 3-kinase activity associated with IRS-1 was also reduced in cells overexpressing calmodulin, while this activity was increased in cells incubated with the cell-permeable calmodulin antagonist trifluoperazine. These data demonstrate an enhanced interaction between calmodulin and IRS proteins in models of insulin resistance and suggest a possible mechanism by which increased intracellular Ca(2+) concentrations may contribute to impaired insulin sensitivity.

Functional interactions between the p35 subunit of the Arp2/3 complex and calmodulin in yeast.

The end9-1 (arc35-1) mutant was identified as an endocytosis mutant and is a mutant allele of ARC35 that encodes a subunit of the Arp2/3 complex. As for other mutants in the Arp2/3 complex, arc35-1 is defective for endocytosis and organization of the actin cytoskeleton. Both defects can be suppressed by overexpression of calmodulin. Analysis of a collection of temperature-sensitive cmd1 mutants for their ability to suppress either the endocytic defect and/or the actin defect indicates that the two defects are tightly coupled. We demonstrate that Arc35p and Cmd1p interact and that Arc35p is required for cortical localization of calmodulin. This is the first report linking Arp2/3 complex function with calmodulin through which it exercises at least one of its endocytic functions.