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Aims c-jun N-terminal kinase (JNK) is a critical stress response kinase that activates in a wide range of physiological and pathological cellular processes. We recently discovered a pivotal role of JNK in the development of atrial arrhythmias in the aged heart, while cardiac CaMKIIδ, another pro-arrhythmic molecule, was also known to enhance atrial arrhythmogenicity. Here, we aimed to reveal a regulatory role of the stress kinase JNK2 isoform on CaMKIIδ expression. Methods and results Activated JNK2 leads to increased CaMKIIδ protein expression in aged human and mouse atria, evidenced from the reversal of CaMKIIδ up-regulation in JNK2 inhibitor treated wild-type aged mice. This JNK2 action in CaMKIIδ expression was further confirmed in HL-1 myocytes co-infected with AdMKK7D-JNK2, but not when co-infected with AdMKK7D-JNK1. JNK2-specific inhibition (either by a JNK2 inhibitor or overexpression of inactivated dominant-negative JNK2 (JNK2dn) completely attenuated JNK activator anisomycin-induced CaMKIIδ up-regulation in HL-1 myocytes, whereas overexpression of JNK1dn did not. Moreover, up-regulated CaMKIIδ mRNA along with substantially increased phosphorylation of JNK downstream transcription factor c-jun [but not activating transcription factor2 (ATF2)] were exhibited in both aged atria (humans and mice) and transiently JNK activated HL-1 myocytes. Cross-linked chromatin-immunoprecipitation assays (XChIP) revealed that both c-jun and ATF2 were bound to the CaMKIIδ promoter, but significantly increased binding of c-jun only occurred in the presence of anisomycin and JNK inhibition alleviated this anisomycin-elevated c-jun binding. Mutated CaMKII consensus c-jun binding sites impaired its promoter activity. Enhanced transcriptional activity of CaMKIIδ by anisomycin was also completely reversed to the baseline by either JNK2 siRNA or c-jun siRNA knockdown. Conclusion JNK2 activation up-regulates CaMKIIδ expression in the aged atrium. This JNK2 regulation in CaMKIIδ expression occurs at the transcription level through the JNK downstream transcription factor c-jun. The discovery of this novel molecular mechanism of JNK2-regulated CaMKII expression sheds new light on possible anti-arrhythmia drug development.

Calcium (Ca(2+)) signaling within the cell nucleus regulates specific cellular events such as gene transcription and cell proliferation. Nuclear and cytosolic Ca(2+) levels can be independently regulated, and nuclear translocation of receptor tyrosine kinases (RTKs) is one way to locally activate signaling cascades within the nucleus. Nuclear RTKs, including the epidermal growth factor receptor (EGFR), are important for processes such as transcriptional regulation, DNA-damage repair, and cancer therapy resistance. RTKs can hydrolyze phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) within the nucleus, leading to Ca(2+) release from the nucleoplasmic reticulum by inositol 1,4,5-trisphosphate receptors. PI(4,5)P(2) hydrolysis is mediated by phospholipase C (PLC). However, it is unknown which nuclear PLC isoform is triggered by EGFR. Here, using subcellular fractionation, immunoblotting and fluorescence, siRNA-based gene knockdowns, and FRET-based biosensor reporter assays, we investigated the role of PLCδ4 in epidermal growth factor (EGF)-induced nuclear Ca(2+) signaling and downstream events. We found that EGF-induced Ca(2+) signals are inhibited when translocation of EGFR is impaired. Nuclear Ca(2+) signals also were reduced by selectively buffering inositol 1,4,5-trisphosphate (InsP(3)) within the nucleus. EGF induced hydrolysis of nuclear PI(4,5)P(2) by the intranuclear PLCδ4, rather than by PLCγ1. Moreover, protein kinase C, a downstream target of EGF, was active in the nucleus of stimulated cells. Furthermore, PLCδ4 and InsP(3) modulated cell cycle progression by regulating the expression of cyclins A and B1. These results provide evidence that EGF-induced nuclear signaling is mediated by nuclear PLCδ4 and suggest new therapeutic targets to modulate the proliferative effects of this growth factor.

In heart, the type 2 inositol 1,4,5-triphosphate receptor (InsP3R2) is the predominant isoform expressed and is localized in the nuclear membrane of ventricular myocytes. InsP3R2-mediated Ca(2+) release regulates hypertrophy specific gene expression by modulating CaMKIIδ, histone deacetylase, and calcineurin-NFATc signaling pathways. InsP3R2 protein is a hypertrophy specific marker and is overexpressed in heart failure animal models and in humans. However, the regulation of InsP3R2 mRNA and protein expression during cardiac hypertrophy and heart failure is not known. Here we show the transcriptional regulation of the Itpr2 gene in adult cardiomyocytes. Our data demonstrates that, InsP3R2 mRNA and protein expression is activated by hypertrophic agonists and attenuated by InsP3R inhibitors 2-aminoethoxyldiphenyl borate and xestospongin-C. The Itpr2 promoter is regulated by the calcineurin-NFATc signaling pathway. NFATc1 regulates Itpr2 gene expression by directly binding to the Itpr2 promoter. The calcineurin-NFATc mediated up-regulation of the Itpr2 promoter was attenuated by cyclosporine-A. InsP3R2 mRNA and protein expression was up-regulated in calcineurin-A transgenic mice and in human heart failure. Collectively, our data suggests that ITPR2 and hypertrophy specific gene expression is regulated, in part, by a positive feedback regulation between InsP3R2 and calcineurin-NFATc signaling pathways.

Inositol 1,4,5-trisphosphate (IP3) receptor (IP3R)-dependent Ca2+signaling exerts positive inotropic, but also arrhythmogenic, effects on excitation-contraction coupling (ECC) in the atrial myocardium. The role of IP3R-dependent sarcoplasmic reticulum (SR) Ca2+release in ECC in the ventricular myocardium remains controversial. Here we investigated the role of this signaling pathway during ECC in isolated rabbit ventricular myocytes. Immunoblotting of proteins from ventricular myocytes showed expression of both type 2 and type 3 IP3R at levels ∼3.5-fold less than in atrial myocytes. In permeabilized myocytes, direct application of IP3(10 μM) produced a transient 21% increase in the frequency of Ca2+sparks ( P < 0.05). This increase was accompanied by a 13% decrease in spark amplitude ( P < 0.05) and a 7% decrease in SR Ca2+load ( P < 0.05) and was inhibited by IP3R antagonists 2-aminoethoxydiphenylborate (2-APB; 20 μM) and heparin (0.5 mg/ml). In intact myocytes endothelin-1 (100 nM) was used to stimulate IP3production and caused a 38% ( P < 0.05) increase in the amplitude of action potential-induced (0.5 Hz, field stimulation) Ca2+transients. This effect was abolished by the IP3R antagonist 2-APB (2 μM) or by using adenoviral expression of an IP3affinity trap that buffers cellular IP3. Together, these data suggest that in rabbit ventricular myocytes IP3R-dependent Ca2+release has positive inotropic effects on ECC by facilitating Ca2+release through ryanodine receptor clusters.

In cardiac myocytes the type-2 inositol 1,4,5-trisphosphate receptor (IP(3)R2) is the predominant isoform expressed. The IP(3)R2 channel is localized to the SR and to the nuclear envelope. We studied IP(3)-dependent nuclear Ca(2+) signals ([Ca(2+)](Nuc)) in permeabilized atrial myocytes and in isolated cardiac nuclei. In permeabilized myocytes IP(3) (20 microm) and the more potent IP(3)R agonist adenophostin (5 microm) caused an elevation of [Ca(2+)](Nuc). An IP(3)-dependent increase of [Ca(2+)](Nuc) was still observed after pretreatment with tetracaine to block Ca(2+) release from ryanodine receptors (RyRs), and the effect of IP(3) was partially reversed or prevented by the IP(3)R blockers heparin and 2-APB. Isolated nuclei were superfused with an internal solution containing the Ca(2+) indicator fluo-4 dextran. Exposure to IP(3) (10 microm) and adenophostin (0.5 microm) increased [Ca(2+)](Nuc) by 25 and 27%, respectively. [Ca(2+)](Nuc) increased to higher levels than [Ca(2+)](Cyt) immediately adjacent to the outer membrane of the nuclear envelope, suggesting that a significant portion of nuclear IP(3) receptors are facing the nucleoplasm. When nuclei were pretreated with heparin or 2-APB, IP(3) failed to increase [Ca(2+)](Nuc). Isolated nuclei were also loaded with the membrane-permeant low-affinity Ca(2+) probe fluo-5N AM which compartmentalized into the nuclear envelope. Exposure to IP(3) and adenophostin resulted in a decrease of the fluo-5N signal that could be prevented by heparin. Stimulation of IP(3)R caused depletion of the nuclear Ca(2+) stores by approximately 60% relative to the maximum depletion produced by the ionophores ionomycin and A23187. The fluo-5N fluorescence decrease was particularly pronounced in the nuclear periphery, suggesting that the nuclear envelope may represent the predominant nuclear Ca(2+) store. The data indicate that IP(3) can elicit Ca(2+) release from cardiac nuclei resulting in localized nuclear Ca(2+) signals.

The type 2 inositol 1,4,5-trisphosphate receptor (InsP3R2) was identified previously as the predominant isoform in cardiac ventricular myocytes. Here we reported the subcellular localization of InsP3R2 to the cardiomyocyte nuclear envelope (NE). The other major known endo/sarcoplasmic reticulum calcium-release channel (ryanodine receptor) was not localized to the NE, indicating functional segregation of these channels and possibly a unique role for InsP3R2 in regulating nuclear calcium dynamics. Immunoprecipitation experiments revealed that the NE InsP3R2 associates with Ca2+/calmodulin-dependent protein kinase IIδ (CaMKIIδ), the major isoform expressed in cardiac myocytes. Recombinant InsP3R2 and CaMKIIδB also co-immunoprecipitated after co-expression in COS-1 cells. Additionally, the amino-terminal 1078 amino acids of the InsP3R2 were sufficient for interaction with CaMKIIδB and associated upon mixing following separate expression. CaMKII can also phosphorylate InsP3R2, as demonstrated by 32P labeling. Incorporation of CaMKII-treated InsP3R2 into planar lipid bilayers revealed that InsP3-mediated channel open probability is significantly reduced (∼11 times) by phosphorylation via CaMKII. We concluded that the InsP3R2 and CaMKIIδ likely represent two central components of a multiprotein signaling complex, and this raises the possibility that calcium release via InsP3R2 in the myocyte NE may activate local CaMKII signaling, which may feedback on InsP3R2 function.

In non-excitable cells, the inositol 1,4,5-trisphosphate receptor (IP3R) is an intracellular Ca2+ channel playing a major role in Ca2+ signaling. Three isoforms of IP3R have been identified and most cell types express different proportions of each isoform. The DT40 B lymphocyte cell line lacking all three IP3R isoforms (DT40IP3R-KO cells) represents an excellent model to re-express any recombinant IP3R and analyze its specific properties. In the study presented here, we confirmed that DT40IP3R-KO cells do not express any IP3-sensitive Ca2+ release channel. However, with an immunoblot approach and a [3H]IP3 binding approach we demonstrated the presence of a C-terminally truncated form of IP3R type III in the cytosolic fraction of DT40IP3R-KO cells. We further showed that this truncated IP3R retained the ability to couple to the Ca2+ entry channel TRPC6. Therefore, a word of caution is offered about the interpretation of results obtained in using DT40IP3R-KO cells to study the cellular mechanisms of Ca2+ entry.

In the present study, we examined the mechanisms through which erythropoietin (Epo) activates the calcium-permeable transient receptor potential protein channel (TRPC)2. Erythroblasts were isolated from the spleens of phenylhydrazine-treated mice, and Epo stimulation resulted in a significant and dose-dependent increase in intracellular calcium concentration ([Ca2+]i). This increase in [Ca2+]iwas inhibited by pretreatment with the phospholipase C (PLC) inhibitor U-73122 but not by the inactive analog U-73343, demonstrating the requirement for PLC activity in Epo-modulated Ca2+influx in primary erythroid cells. To determine whether PLC is involved in the activation of TRPC2 by Epo, cell models were used to examine this interaction. Single CHO-S cells that expressed transfected Epo receptor (Epo-R) and TRPC2 were identified, and [Ca2+]iwas quantitated. Epo-induced Ca2+influx through TRPC2 was inhibited by pretreatment with U-73122 or by downregulation of PLCγ1 by RNA interference. PLC activation results in the production of inositol 1,4,5-trisphosphate (IP3), and TRPC2 has IP3receptor (IP3R) binding sites. To determine whether IP3R is involved in Epo-R signaling, TRPC2 mutants were prepared with partial or complete deletions of the COOH-terminal IP3R binding domains. In cells expressing TRPC2 IP3R binding mutants and Epo-R, no significant increase in [Ca2+]iwas observed after Epo stimulation. TRPC2 coassociated with Epo-R, PLCγ, and IP3R, and the association between TRPC2 and IP3R was disrupted in these mutants. Our data demonstrate that Epo-R modulates TRPC2 activation through PLCγ; that interaction of IP3R with TRPC2 is required; and that Epo-R, TRPC2, PLCγ, and IP3R interact to form a signaling complex.

The InsP3R proteins have three recognized domains, the InsP3-binding, regulatory/coupling, and channel domains (Mignery, G.A., and T.C. Südhof. 1990. EMBO J. 9:3893–3898). The InsP3 binding domain and the channel-forming domain are at opposite ends of the protein. Ligand regulation of the channel must involve communication between these different regions of the protein. This communication likely involves the interceding sequence (i.e., the regulatory/coupling domain). The single channel functional attributes of the full-length recombinant type-1, -2, and -3 InsP3R channels have been defined. Here, two type-1/type-2 InsP3R regulatory/coupling domain chimeras were created and their single channel function defined. One chimera (1-2-1) contained the type-2 regulatory/coupling domain in a type-1 backbone. The other chimera (2-1-2) contained the type-1 regulatory/coupling domain in a type-2 backbone. These chimeric proteins were expressed in COS cells, isolated, and then reconstituted in proteoliposomes. The proteoliposomes were incorporated into artificial planar lipid bilayers and the single-channel function of the chimeras defined. The chimeras had permeation properties like that of wild-type channels. The ligand regulatory properties of the chimeras were altered. The InsP3 and Ca2+ regulation had some unique features but also had features in common with wild-type channels. These results suggest that different independent structural determinants govern InsP3R permeation and ligand regulation. It also suggests that ligand regulation is a multideterminant process that involves several different regions of the protein. This study also demonstrates that a chimera approach can be applied to define InsP3R structure-function.

The inositol 1,4,5-trisphosphate receptor (InsP(3)R) is a tetrameric assembly of conserved subunits that each contains six transmembrane regions (TMRs) localized near the carboxyl terminus. Receptor subunit assembly into a tetramer appears to be a multideterminant process involving an additive contribution of membrane spanning helices and the short cytosolic carboxyl terminus (residues 2590-2749). Previous studies have shown that of the six membrane-spanning regions in each subunit, the 5th and 6th transmembrane regions, and the carboxyl terminus are strong determinants for assembly. The fifth and sixth TMRs contain numerous beta-branched amino acids that may participate in coiled/coil formation via putative leucine zipper motifs. InsP(3)R truncation mutants were expressed in COS-1 cells and analyzed by sucrose density gradient sedimentation and gel filtration for their ability to assemble. Chemical cross-linking with the homobifunctional reagents sDST or DMS of mammalian and bacterially expressed carboxyl-terminal containing receptor fragments reveals that sequences within the carboxyl terminus confer the formation of subunit dimers. A series of InsP(3) receptor carboxyl-terminal fragments and glutathione S-transferase (GST)/InsP(3)R chimeras were expressed in Escherichia coli and used in an in vitro assay to elucidate the minimal sequence responsible for association of the carboxyl termini into dimers. The results presented here indicate that this minimal sequence is approximately 30 residues in length and is localized between residues 2629 and 2654. These residues are highly conserved between the three InsP(3)R isoforms ( approximately 80% identity) as well as the ryanodine receptor ( approximately 40% identity) and suggest that a conserved assembly motif may exist between the two intracellular receptor families. We propose that assembly of the InsP(3) receptor to a tetramer involves intersubunit interactions mediated through both the membrane-spanning regions and residues 2629-2654 of the carboxyl terminus possibly through the formation of a dimer of dimers.

Astrocytes respond to neuronal activity by propagating Ca(2+) waves elicited through the inositol 1,4,5-trisphosphate pathway. We have previously shown that wave propagation is supported by specialized Ca(2+) release sites, where a number of proteins, including inositol 1,4,5-trisphosphate receptors (IP(3)R), occur together in patches. The specific IP(3)R isoform expressed by astrocytes in situ in rat brain is unknown. In the present report, we use isoform-specific antibodies to localize immunohistochemically the IP(3)R subtype expressed in astrocytes in rat brain sections. Astrocytes were identified using antibodies against the astrocyte-specific markers, S-100 beta, or GFAP. Dual indirect immunohistochemistry showed that astrocytes in all regions of adult rat brain express only IP(3)R2. High-resolution analysis showed that hippocampal astrocytes are endowed with a highly branched network of processes that bear fine hair-like extensions containing punctate patches of IP(3)R2 staining in intimate contact with synapses. Such an organization is reminiscent of signaling microdomains found in cultured glial cells. Similarly, Bergmann glial cell processes in the cerebellum also contained fine hair-like processes containing IP(3)R2 staining. The IP(3)R2-containing fine terminal branches of astrocyte processes in both brain regions were found juxtaposed to presynaptic terminals containing synaptophysin as well as PSD 95-containing postsynaptic densities. Corpus callosum astrocytes had an elongated morphology with IP(3)R2 studded processes extending along fiber tracts. Our data suggest that PLC-mediated Ca(2+) signaling in astrocytes in rat brain occurs predominantly through IP(3)R2 ion channels. Furthermore, the anatomical arrangement of the terminal astrocytic branches containing IP(3)R2 ensheathing synapses is ideal for supporting glial monitoring of neuronal activity.

Fast two-dimensional confocal microscopy and the Ca(2+) indicator fluo-4 were used to study excitation-contraction (E-C) coupling in cat atrial myocytes which lack transverse tubules and contain both subsarcolemmal junctional (j-SR) and central nonjunctional (nj-SR) sarcoplasmic reticulum. Action potentials elicited by field stimulation induced transient increases of intracellular Ca(2+) concentration ([Ca(2+)](i)) that were highly inhomogeneous. Increases started at distinct subsarcolemmal release sites spaced approximately 2 microm apart. The amplitude and the latency of Ca(2+) release from these sites varied from beat to beat. Subsarcolemmal release fused to build a peripheral ring of elevated [Ca(2+)](i), which actively propagated to the center of the cells via Ca(2+)-induced Ca(2+) release. Resting myocytes exhibited spontaneous Ca(2+) release events, including Ca(2+) sparks and local (microscopic) or global (macroscopic) [Ca(2+)](i) waves. The microscopic [Ca(2+)](i) waves propagated in a saltatory fashion along the sarcolemma ("coupled" Ca(2+) sparks) revealing the sequential activation of Ca(2+) release sites of the j-SR. Moreover, during global [Ca(2+)](i) waves, Ca(2+) release was evident from individual nj-SR sites. Ca(2+) release sites were arranged in a regular three-dimensional grid as deduced from the functional data and shown by immunostaining of ryanodine receptor Ca(2+) release channels. The longitudinal and transverse distances between individual Ca(2+) release sites were both approximately 2 microm. Furthermore, electron microscopy revealed a continuous sarcotubular network and one peripheral coupling of j-SR with the sarcolemma per sarcomere. The results demonstrate directly that, in cat atrial myocytes, the action potential-induced whole-cell [Ca(2+)](i) transient is the spatio-temporal summation of Ca(2+) release from subsarcolemmal and central sites. First, j-SR sites are activated in a stochastic fashion by the opening of voltage-dependent sarcolemmal Ca(2+) channels. Subsequently, nj-SR sites are activated by Ca(2+)-induced Ca(2+) release propagating from the periphery.

A full-length rat type 2 inositol 1,4,5-trisphosphate (InsP(3)) receptor cDNA construct was generated and expressed in COS-1 cells. Targeting of the full-length recombinant type 2 receptor protein to the endoplasmic reticulum was confirmed by immunocytochemistry using isoform specific affinity-purified antibodies and InsP(3)R-green fluorescent protein chimeras. The receptor protein was solubilized and incorporated into proteoliposomes for functional characterization. Single-channel recordings from proteoliposomes fused into planar lipid bilayers revealed that the recombinant protein formed InsP(3)- and Ca(2+)-sensitive ion channels. The unitary conductance ( approximately 250 pS; 220/20 mM Cs(+) as charge carrier), gating, InsP(3), and Ca(2+) sensitivities were similar to those previously described for the native type 2 InsP(3)R channel. However, the maximum open probability of the recombinant channel was slightly lower than that of its native counterpart. These data show that our full-length rat type 2 InsP(3)R cDNA construct encodes a protein that forms an ion channel with functional attributes like those of the native type 2 InsP(3)R channel. The possibility of measuring the function of single recombinant type 2 InsP(3)R is a significant step toward the use of molecular tools to define the determinants of isoform-specific InsP(3)R function and regulation.

The inositol 1,4,5-trisphosphate receptor (InsP3R) is a tetrameric assembly of highly conserved subunits that contain multiple membrane-spanning sequences in the C-terminal region of the protein. In studies aimed at investigating the oligomerization and transmembrane topology of the type-1 InsP3R, a series of membrane-spanning region truncation and deletion plasmids were constructed. These plasmids were transiently transfected in COS-1 cells, and the resulting expression products were analyzed for the ability to assemble into tetrameric structures. The topology of the membrane-spanning region truncations and the full-length receptor was determined by immunocytochemical analysis of transfected COS-1 cells using complete or selective permeabilization strategies. Our results are the first to experimentally define the presence of six membrane-spanning regions. These results are consistent with the current model for the organization of the InsP3R in the endoplasmic reticulum and show that the truncation mutants are properly targeted and oriented in the endoplasmic reticulum membrane, thus making them amenable reagents to study receptor subunit oligomerization. Fractionation of soluble and membrane protein components revealed that the first two membrane-spanning regions were necessary for membrane targeting of the receptor. Sedimentation and immunoprecipitation experiments show that assembly of the receptor subunits was an additive process as the number of membrane-spanning regions increased. Immunoprecipitations from cells co-expressing the full-length receptor and carboxyl-terminal truncations reveal that constructs expressing the first two or more membrane-spanning domains were capable of co-assembling with the full-length receptor. Inclusion of the fifth membrane-spanning segment significantly enhanced the degree of oligomerization. Furthermore, a deletion construct containing only membrane-spanning regions 5 and 6 oligomerized to a similar extent as that of the wild type protein. Membrane-spanning region deletion constructions that terminate with the receptor’s 145 carboxyl-terminal amino acids were found to have enhanced assembly characteristics and implicate the carboxyl terminus as a determinant in oligomerization. Our results reveal a process of receptor assembly involving several distinct yet additive components and define the fifth and sixth membrane spanning regions as the key determinants in receptor oligomerization.

The inositol 1,4,5-trisphosphate receptor (InsP3R) forms ligand-regulated intracellular Ca2+ release channels in the endoplasmic reticulum of all mammalian cells. The InsP3R has been suggested to have six transmembrane regions (TMRs) near its carboxyl terminus. A TMR-deletion mutation strategy was applied to define the location of the InsP3R pore. Mutant InsP3Rs were expressed in COS-1 cells and single channel function was defined in planar lipid bilayers. Mutants having the fifth and sixth TMR (and the interceding lumenal loop), but missing all other TMRs, formed channels with permeation properties similar to wild-type channels (gCs = 284; gCa = 60 pS; PCa/PCs = 6.3). These mutant channels bound InsP3, but ligand occupancy did not regulate the constitutively open pore (Po > 0.80). We propose that a region of 191 amino acids (including the fifth and sixth TMR, residues 2398–2589) near the COOH terminus of the protein forms the InsP3R pore. Further, we have produced a constitutively open InsP3R pore mutant that is ideal for future site-directed mutagenesis studies of the structure–function relationships that define Ca2+ permeation through the InsP3R channel.

The functional role of inositol 1,4,5-trisphosphate (InsP3) signaling in cardiomyocytes is not entirely understood but it was linked to an increased propensity for triggered activity. The aim of this study was to determine how InsP3 receptors can translate Ca(2+) release into a depolarization of the plasma membrane and consequently arrhythmic activity. We used embryonic stem cell-derived cardiomyocytes (ESdCs) as a model system since their spontaneous electrical activity depends on InsP3-mediated Ca(2+) release. [InsP3]i was monitored with the FRET-based InsP3-biosensor FIRE-1 (Fluorescent InsP3 Responsive Element) and heterogeneity in sub-cellular [InsP3]i was achieved by targeted expression of FIRE-1 in the nucleus (FIRE-1nuc) or expression of InsP3 5-phosphatase (m43) localized to the plasma membrane. Spontaneous activity of ESdCs was monitored simultaneously as cytosolic Ca(2+) transients (Fluo-4/AM) and action potentials (current clamp). During diastole, the diastolic depolarization was paralleled by an increase of [Ca(2+)]i and spontaneous activity was modulated by [InsP3]i. A 3.7% and 1.7% increase of FIRE-1 FRET ratio and 3.0 and 1.5 fold increase in beating frequency was recorded upon stimulation with endothelin-1 (ET-1, 100 nmol/L) or phenylephrine (PE, 10 µmol/L), respectively. Buffering of InsP3 by FIRE-1nuc had no effect on the basal frequency while attenuation of InsP3 signaling throughout the cell (FIRE-1), or at the plasma membrane (m43) resulted in a 53.7% and 54.0% decrease in beating frequency. In m43 expressing cells the response to ET-1 was completely suppressed. Ca(2+) released from InsP3Rs is more effective than Ca(2+) released from RyRs to enhance INCX. The results support the hypothesis that in ESdCs InsP3Rs form a functional signaling domain with NCX that translates Ca(2+) release efficiently into a depolarization of the membrane potential.

The inositol 1,4,5-trisphosphate receptor (InsP3R) is an intracellular Ca2+ release channel that mediates the rise in cytoplasmic calcium in response to receptor-activated production of InsP3. The InsP3R-mediated signaling pathway appears to be ubiquitous and is involved in many cellular processes including cell division, smooth muscle contraction, and neuronal signaling. Different regions of the heart also express InsP3 receptors. We report here that acutely dissociated ventricular myocytes from ferret and rat hearts express significant levels of InsP3R as indicated by immunoblotting with a receptor consensus antibody. InsP3binding experiments (K D = 23.6 nm andB max = 0.46 pmol/mg) suggest the myocytes contain the high affinity type 2 InsP3 receptor. Exhaustive mRNA screening by polymerase chain reaction, RNase protection, and subsequent DNA sequencing positively identify the InsP3R as type 2. The type 2 receptor from ferret heart was then incorporated into planar lipid bilayers and formed Ca2+-selective, InsP3-activated, heparin-blocked ion channels. We conclude that the predominant InsP3 receptor isoform expressed in cardiac myocytes is type 2 and that it forms a functional InsP3-gated Ca2+ channel when reconstituted in planar lipid bilayers.

We previously reported in atrial myocytes that inhibition of cAMP-dependent protein kinase (PKA) by laminin (LMN)-integrin signaling activates β2-adrenergic receptor (β2-AR) stimulation of cytosolic phospholipase A2 (cPLA2). The present study sought to determine the signaling mechanisms by which inhibition of PKA activates β2-AR stimulation of cPLA2. We therefore determined the effects of zinterol (0.1 μM; zint-β2-AR) to stimulate ICa,L in atrial myocytes in the absence (+PKA) and presence (-PKA) of the PKA inhibitor (1 μM) KT5720 and compared these results with atrial myocytes attached to laminin (+LMN). Inhibition of Raf-1 (10 μM GW5074), phospholipase C (PLC; 0.5 μM edelfosine), PKC (4 μM chelerythrine) or IP3 receptor (IP3R) signaling (2 μM 2-APB) significantly inhibited zint-β2-AR stimulation of ICa,L in-PKA but not +PKA myocytes. Western blots showed that zint-β2-AR stimulation increased ERK1/2 phosphorylation in-PKA compared to +PKA myocytes. Adenoviral (Adv) expression of dominant negative (dn) -PKCα, dn-Raf-1 or an IP3 affinity trap, each inhibited zint-β2-AR stimulation of ICa,L in + LMN myocytes compared to control +LMN myocytes infected with Adv-βgal. In +LMN myocytes, zint-β2-AR stimulation of ICa,L was enhanced by adenoviral overexpression of wild-type cPLA2 and inhibited by double dn-cPLA2S505A/S515A mutant compared to control +LMN myocytes infected with Adv-βgal. In-PKA myocytes depletion of intracellular Ca2+ stores by 5 μM thapsigargin failed to inhibit zint-β2-AR stimulation of ICa,L via cPLA2. However, disruption of caveolae formation by 10 mM methyl-β-cyclodextrin inhibited zint-β2-AR stimulation of ICa,L in-PKA myocytes significantly more than in +PKA myocytes. We conclude that inhibition of PKA removes inhibition of Raf-1 and thereby allows β2-AR stimulation to act via PKCα/Raf-1/MEK/ERK1/2 and IP3-mediated Ca2+ signaling to stimulate cPLA2 signaling within caveolae. These findings may be relevant to the remodeling of β-AR signaling in failing and/or aging heart, both of which exhibit decreases in adenylate cyclase activity.

InsP(3)-mediated calcium release through the type 2 inositol 1,4,5-trisphosphate receptor (InsP(3)R2) in cardiac myocytes results in the activation of associated CaMKII, thus enabling the kinase to act on downstream targets, such as histone deacetylases 4 and 5 (HDAC4 and HDAC5). The CaMKII activity also feedback modulates InsP(3)R2 function by direct phosphorylation and results in a dramatic decrease in the receptor-channel open probability (P(o)). We have identified S150 in the InsP(3)R2 core suppressor domain (amino acids 1-225) as the specific residue that is phosphorylated by CaMKII. Site-directed mutagenesis reveals that S150 is the CaMKII phosphorylation site responsible for modulation of channel activity. Nonphosphorylatable (S150A) and phosphomimetic (S150E) mutations were studied in planar lipid bilayers. The InsP(3)R2 S150A channel showed no decrease in activity when treated with CaMKII. Conversely, the phosphomimetic (S150E) channel displayed a very low P(o) under normal recording conditions in the absence of CaMKII (2 μM InsP(3) and 250 nM [Ca(2+)](FREE)) and mimicked a WT channel that has been phosphorylated by CaMKII. Phopho-specific antibodies demonstrate that InsP(3)R2 Ser-150 is phosphorylated in vivo by CaMKIIδ. The results of this study show that serine 150 of the InsP(3)R2 is phosphorylated by CaMKII and results in a decrease in the channel open probability.

Inositol 1,4,5-triphosphosphate receptors are a family of intracellular Ca2+ channels, modulated by the ligands InsP3 and Ca2+ in response to an array of neuro-hormonal stimuli to release Ca2+ from internal stores. In cardiac myocytes, the InsP3R2, CaMKII and CaM are components of a macromolecular signalplex. However, modulation of InsP3R’s Ca2+ channel and the underlying mechanism(s) that converge and integrate nuclear hypertrophic signals to initiate transcription is not fully established. Here, we delineate the mechanistic regulation of nuclear InsP3R2’s bidirectional Ca2+ channel activity and its effect on the activation of nuclear hypertrophic signaling. We show that, endothelin-1 (ET1) stimulates elevated InsP3 induced Ca2+ release into the nucleus from perinuclear InsP3 receptors. This elevated nuclear Ca2+ release is due to CaMKIIδ mediated phosphorylation of cytoplasmic oriented InsP3R2 inhibiting Ca2+ release to the cytoplasm. This increase in nuclear Ca2+ activates Ca2+ sensitive transctiption factors thereby activating gene expression. The elevated nuclear Ca2+ and gene expression can be attenuated by CaMKII inhibitor KN-93 and InsP3R antagonist 2-ABP. We also show that, exogenous InsP3R2 was phosphorylated by both cytoplasmic (CaMKIIδC) and nuclear (CaMKIIδB) isoforms of CaMKIIδ. Intriguingly, CaMKIIδC phosphorylates endogenous InsP3R2 in cardiomyocytes more efficiently than CaMKIIδB and not by its kinase-dead derivatives. In conclusion, our findings demonstrate that, InsP3R2 mediated Ca2+ signaling integrates and vectors the GPCR activated hypertrophic signals to the nucleus triggering gene transcription underlying cardiac hypertrophy.

Although agonist stimulation leads to an increase in inositol 1,4,5-trisphosphate (InsP3) and decreased calcium in peripherally and centrally located sarcoplasmic reticulum in smooth muscle, the distribution of InsP3 receptors is unknown. InsP3 receptor and the calcium binding protein, calsequestrin were localized by immunolabelling in a tonic and a phasic smooth muscle. InsP3 receptor labelling was predominantly localized at the cell periphery, where most of the sarcoplasmic reticulum is localized in vas deferens (phasic muscle). Elements of central sarcoplasmic reticulum, where present, were also labelled. Distribution of calsequestrin in vas deferens was similar to that of the InsP3 receptor. In aorta (tonic muscle) the InsP3 receptor labelling was proportional to sarcoplasmic reticulum distribution: predominantly central. No labelling of sections or immunoblots was observed with the anti-calsequestrin antibody in aorta. InsP3 and caffeine, but not cyclic ADP-ribose, released intracellular Ca2+ in permeabilized vas deferens and aorta. The ultrastructure of the sarcoplasmic reticulum, investigated in stereo views of semi-thick and thin sections of osmium ferricyanide stained tissue, is shown to have several distinctive features, such as fenestrated sheets (single or in stacks), as well as numerous regions of continuity between central and peripheral sarcoplasmic reticulum, suggesting a single compartment within the smooth muscle cell. Regions of the sarcoplasmic reticulum were closely apposed to and often ensheathed mitochondria. We conclude that InsP3 receptors are present in both the central and the peripheral sarcoplasmic reticulum of tonic and phasic smooth muscle, consistent with electron probe analysis results showing calcium release from both regions.

Inositol 1,4,5-trisphosphate (InsP3) is a ubiquitous second messenger in eukaryotic cells that triggers Ca2+ release from intracellular stores. Three types of InsP3 receptors have been identified in mammals. The three receptor types are encoded by homologous genes and are structurally similar, suggesting two alternative hypotheses about the biological significance of multiple InsP3 receptors: (a) the different InsP3 receptors could have similar functions as InsP3-gated Ca2+ channels, and the presence of multiple genes could then serve as a mechanism to allow tissue-specific differential expression of receptors; or (b) the different receptors are co-expressed in cells but have distinct biological roles in these cells. To test these hypothesis, we have investigated the similarities and differences between the expression, alternative splicing, and ligand binding of different receptors. Our results demonstrate co-expression of different InsP3 receptors in almost all tissues and cell lines tested. Although all receptor types exhibit a similar specificity for inositol phosphates, the different receptors have different affinities for InsP3, with a relative order of affinities of type II > type I > type III. These findings suggest that the presence of multiple InsP3-sensitive Ca2+ pools with differential responsiveness to InsP3 may be a general property of all cells mediated by the presence of multiple types of InsP3 receptors.

The inositol 1,4,5-trisphosphate receptor (IP3R) is expressed at very high levels in cerebellar Purkinje cells. Within these neurons, it has a widespread distribution throughout the endoplasmic reticulum (ER) and is present at particularly high concentrations at sites of membrane appositions within peculiar stacks of ER cisternae. Here we report that stacks of ER cisternae, reminiscent of those observed in Purkinje cells, can be induced by overexpression of full-length IP3R, but not of mutant forms of the protein in COS cells. Within these stacks the IP3R forms a crystalline array at apposed cisternal faces. Additionally, we show that Purkinje cell stacks are not permanent structures. Our findings suggest that massive stack formation in purkinje cells represents an adaptive response of the ER to hypoxic conditions and is due to the presence of the high concentration of IP3R in its membranes.

Ca2+ efficiently inhibits binding of inositol 1,4,5-trisphosphate (InsP3) to the InsP3 receptor in cerebellar membranes but not to the purified receptor. We have now investigated the mechanism of action by which Ca2+ inhibits InsP3 binding. Our results suggest that Ca2+ does not cause the stable association of a Ca(2+)-binding protein with the receptor. Instead, Ca2+ leads to the production of a soluble, heat-stable, low molecular weight substance from cerebellar membranes that competes with InsP3 for binding. This inhibitory substance probably represents endogenously generated InsP3 as judged by the fact that it co-purifies with InsP3 on anion-exchange chromatography, competes with [3H]InsP3 binding in a pattern similar to unlabeled InsP3, and is in itself capable of releasing 45Ca2+ from permeabilized cells. A potent Ca(2+)-activated phospholipase C activity producing InsP3 was found in cerebellar microsomes that exhibited a Ca2+ dependence identical to the Ca(2+)-dependent inhibition of InsP3 binding. Together these results suggest that the Ca(2+)-dependent inhibition of InsP3 binding to the cerebellar receptor is due to activation of a Ca(2+)-sensitive phospholipase C enriched in cerebellum. Nevertheless, Ca2+ probably also modulates the InsP3 receptor function by a direct interaction with the receptor that does not affect InsP3 binding but regulates InsP3-dependent channel gating.

Although the presence of a BH4 domain distinguishes the antiapoptotic protein Bcl-2 from its proapoptotic relatives, little is known about its function. BH4 deletion converts Bcl-2 into a proapoptotic protein, whereas a TAT-BH4 fusion peptide inhibits apoptosis and improves survival in models of disease due to accelerated apoptosis. Thus, the BH4 domain has antiapoptotic activity independent of full-length Bcl-2. Here we report that the BH4 domain mediates interaction of Bcl-2 with the inositol 1,4,5-trisphosphate (IP3) receptor, an IP3-gated Ca 2+ channel on the endoplasmic reticulum (ER). BH4 peptide binds to the regulatory and coupling domain of the IP3 receptor and inhibits IP3-dependent channel opening, Ca 2+ release from the ER, and Ca 2+ -mediated apoptosis. A peptide inhibitor of Bcl-2-IP3 receptor interaction prevents these BH4-mediated effects. By inhibiting proapoptotic Ca 2+ signals at their point of origin, the Bcl-2 BH4 domain has the facility to block diverse pathways through which Ca 2+ induces apoptosis.

rab3A is a small neuronal GTP-binding protein specifically localized to synaptic vesicles. Membrane-bound rab3A behaves like an intrinsic membrane protein in vitro, but reversibly dissociates from synaptic vesicles after exocytosis in vivo. Here we demonstrate that rab3A is attached to synaptic vesicle membranes by a carboxy-terminal Cys-X-Cys sequence that is posttranslationally modified. This modification is inhibited by compactin in a mevalonate-dependent manner, suggesting that the Cys-X-Cys sequence represents a novel polyisoprenylation sequence. Isolation of a rab3 homolog from D. melanogaster reveals high evolutionary conservation of rab3A, including its carboxy-terminal Cys-X-Cys sequence. The posttranslational modifications of soluble and membrane-bound rab3A are biochemically different, but both require the carboxy-terminal Cys-X-Cys sequence and are faithfully reproduced in nonneuronal cells. Our results suggest that the carboxy-terminal Cys-X-Cys sequence of rab3A is polyisoprenylated and is used as its regulatable membrane anchor. Furthermore, the hydrophobic modification of rab3A and its correct intracellular targeting to synaptic vesicles are independent, presumably consecutive events.

The inositol-1,4,5-triphosphate (InsP3) receptor consists of a homotetramer of highly conserved 313 kd subunits that contain multiple transmembrane regions in the C-terminal part of the protein. The receptor was expressed in COS cells and its domain structure was studied by mutagenesis. Deletion of the transmembrane regions from the receptor results in the synthesis of a soluble receptor protein that efficiently binds InsP3 but which instead of associating into homotetramers remains monomeric. This result suggests a role for the transmembrane regions in the association of the receptor subunits into tetramers but not in ligand binding. To localize the ligand binding site, further cDNAs encoding truncated receptor proteins were constructed. Assays of InsP3 binding to these truncated InsP3 receptors revealed that sequences in the N-terminal fourth of the InsP3 receptor are sufficient for ligand binding. Accordingly, each subunit of the InsP3 receptor homotetramer contains an independent ligand binding site that is located on the N-terminal ends of each subunit and is separated from the putative channel-forming transmembrane regions by greater than 1400 amino acids. Gel filtration experiments demonstrate a large conformational change of the receptor as a function of ligand binding, suggesting a mechanism by which ligand binding might cause channel opening.

The complete primary structure of the inositol 1,4,5-trisphosphate receptor from rat brain was elucidated using a series of overlapping cDNA clones. Two different sets of clones that either contain or lack a 45-nucleotide sequence in the amino-terminal third of the protein were isolated, suggesting a differential splicing event that results in the biosynthesis of either a 2734- or 2749-amino acid receptor protein. Hydrophobicity analysis demonstrates the presence of a cluster of hydrophobic sequences in the carboxyl-terminal third of the protein that probably comprise eight transmembrane regions and that may form the calcium channel intrinsic to the receptor. The receptor was universally expressed at low levels in all tissues and cultured cells tested. Transfection of a full-length expression construct of the inositol 1,4,5-trisphosphate receptor into COS cells resulted in the biosynthesis of a 260-kDa protein that bound inositol 1,4,5-trisphosphate and formed high molecular weight complexes similar to the native receptor as analyzed by sucrose gradient centrifugations. On the other hand, the protein product synthesized by a mutant receptor construct in which the amino-terminal 418 amino acids were deleted failed to bind inositol 1,4,5-trisphosphate. The mutant receptor still formed high molecular weight complexes, suggesting that it folded normally and that the amino-terminal sequences of the receptor are part of the ligand binding domain.

Neurotransmitters are released at synapses by the Ca2(+)-regulated exocytosis of synaptic vesicles, which are specialized secretory organelles that store high concentrations of neurotransmitters. The rapid Ca2(+)-triggered fusion of synaptic vesicles is presumably mediated by specific proteins that must interact with Ca2+ and the phospholipid bilayer. We now report that the cytoplasmic domain of p65, a synaptic vesicle-specific protein that binds calmodulin contains an internally repeated sequence that is homologous to the regulatory C2-region of protein kinase C (PKC). The cytoplasmic domain of recombinant p65 binds acidic phospholipids with a specificity indicating an interaction of p65 with the hydrophobic core as well as the headgroups of the phospholipids. The binding specificity resembles PKC, except that p65 also binds calmodulin, placing the C2-regions in a context of potential Ca2(+)-regulation that is different from PKC. This is a novel homology between a cellular protein and the regulatory domain of protein kinase C. The structure and properties of p65 suggest that it may have a role in mediating membrane interactions during synaptic vesicle exocytosis.

The antiapoptotic protein Bcl-2 inhibits Ca2+ release from the endoplasmic reticulum (ER). One proposed mechanism involves an interaction of Bcl-2 with the inositol 1,4,5-trisphosphate receptor (IP3R) Ca2+ channel localized with Bcl-2 on the ER. Here we document Bcl-2-IP3R interaction within cells by FRET and identify a Bcl-2 interacting region in the regulatory and coupling domain of the IP3R. A peptide based on this IP3R sequence displaced Bcl-2 from the IP3R and reversed Bcl-2-mediated inhibition of IP3R channel activity in vitro, IP3-induced ER Ca2+ release in permeabilized cells, and cell-permeable IP3 ester-induced Ca2+ elevation in intact cells. This peptide also reversed Bcl-2's inhibition of T cell receptor-induced Ca2+ elevation and apoptosis. Thus, the interaction of Bcl-2 with IP3Rs contributes to the regulation of proapoptotic Ca2+ signals by Bcl-2, suggesting the Bcl-2-IP3R interaction as a potential therapeutic target in diseases associated with Bcl-2's inhibition of cell death.

During cell cycle progression, somatic cells exhibit different patterns of intracellular Ca2+signals during the G0phase, the transition from G1to S, and from G2to M. Because pluripotent embryonic stem (ES) cells progress through cell cycle without the gap phases G1and G2, we aimed to determine whether mouse ES (mES) cells still exhibit characteristic changes of intracellular Ca2+concentration during cell cycle progression. With confocal imaging of the Ca2+-sensitive dye fluo-4 AM, we identified that undifferentiated mES cells exhibit spontaneous Ca2+oscillations. In control cultures where 50.4% of the cells reside in the S phase of the cell cycle, oscillations appeared in 36% of the cells within a colony. Oscillations were not initiated by Ca2+influx but depended on inositol 1,4,5-trisphosphate (IP3)-mediated Ca2+release and the refilling of intracellular stores by a store-operated Ca2+influx (SOC) mechanism. Using cell cycle synchronization, we determined that Ca2+oscillations were confined to the G1/S phase (∼70% oscillating cells vs. G2/M with ∼15% oscillating cells) of the cell cycle. ATP induced Ca2+oscillations, and activation of SOC could be induced in G1/S and G2/M synchronized cells. Intracellular Ca2+stores were not depleted, and all three IP3receptor isoforms were present throughout the cell cycle. Cell cycle analysis after EGTA, BAPTA-AM, 2-aminoethoxydiphenyl borate, thapsigargin, or U-73122 treatment emphasized that IP3-mediated Ca2+release is necessary for cell cycle progression through G1/S. Because the IP3receptor sensitizer thimerosal induced Ca2+oscillations only in G1/S, we propose that changes in IP3receptor sensitivity or basal levels of IP3could be the basis for the G1/S-confined Ca2+oscillations.

During action potential conduction, the axonal specializations at the node, together with the adjacent paranodal terminations of the myelin sheath, interact with glial processes that invest the nodal gap. The nature of the mutual signals between axons and myelinating glia, however, are not well understood. Here we have characterized the distribution of inositol 1,4,5-trisphosphate receptors (IP(3)Rs) in the axoglial apparatus by immunohistochemistry, using known myelin domain-specific markers. While IP(3)R1 is not expressed in the Schwann cells or the axon, IP(3)R2 and IP(3)R3 are expressed in distinct cellular domains, suggesting distinct signaling roles for the two receptors. IP(3)R3 is the most predominant isoform in Schwann cells, and is expressed in particularly dense patches in the paranodal region. In addition to IP(3)Rs, two other members of the metabotropic Ca(2+) signaling pathway, G(alpha)q, and P(2)Y1 type of purinoceptors were also found in Schwann cells. Their pattern of expression matches the expression of their signaling partners, the IP(3)Rs. One interesting finding to emerge from this study is the expression of connexin 32 (Cx32) in close proximity with IP(3)R3. Although IP(3)R3 and Cx32 are not colocalized, their expression in the same membrane areas raises the question whether Schwann cell Ca(2+) signals either control the function of the gap junctions, or whether the gap junctional channels serve as conduits for rapid radial spread of Ca(2+) signals initiated during action potential propagation.

Several lines of evidence indicate that increases in nuclear Ca(2+) have specific biological effects that differ from those of cytosolic Ca(2+), suggesting that they occur independently. The mechanisms involved in controlling nuclear Ca(2+) signaling are both controversial and still poorly understood. Using hypotonic shock combined with mechanical disruption, we obtained and characterized a fraction of purified nuclei from cultured rat skeletal myotubes. Both immunoblot studies and radiolabeled inositol 1,4,5-trisphosphate [IP(3)] binding revealed an important concentration of IP(3) receptors in the nuclear fraction. Immunofluorescence and immunoelectron microscopy studies localized type-1 and type-3 IP(3) receptors in the nucleus with type-1 receptors preferentially localized in the inner nuclear membrane. Type-2 IP(3) receptor was confined to the sarcoplasmic reticulum. Isolated nuclei responded to IP(3) with rapid and transient Ca(2+) concentration elevations, which were inhibited by known blockers of IP(3) signals. Similar results were obtained with isolated nuclei from the 1B5 cell line, which does not express ryanodine receptors but releases nuclear Ca(2+) in an IP(3)-dependent manner. Nuclear Ca(2+) increases triggered by IP(3) evoked phosphorylation of cAMP response element binding protein with kinetics compatible with sequential activation. These results support the idea that Ca(2+) signals, mediated by nuclear IP(3) receptors in muscle cells, are part of a distinct Ca(2+) release component that originates in the nucleus and probably participates in gene regulation mediated by cAMP response element binding protein.

The type 2 inositol 1,4,5-trisphosphate receptor (InsP(3)R2) was identified previously as the predominant isoform in cardiac ventricular myocytes. Here we reported the subcellular localization of InsP(3)R2 to the cardiomyocyte nuclear envelope (NE). The other major known endo/sarcoplasmic reticulum calcium-release channel (ryanodine receptor) was not localized to the NE, indicating functional segregation of these channels and possibly a unique role for InsP(3)R2 in regulating nuclear calcium dynamics. Immunoprecipitation experiments revealed that the NE InsP(3)R2 associates with Ca(2+)/calmodulin-dependent protein kinase IIdelta (CaMKIIdelta), the major isoform expressed in cardiac myocytes. Recombinant InsP(3)R2 and CaMKIIdelta(B) also co-immunoprecipitated after co-expression in COS-1 cells. Additionally, the amino-terminal 1078 amino acids of the InsP(3)R2 were sufficient for interaction with CaMKIIdelta(B) and associated upon mixing following separate expression. CaMKII can also phosphorylate InsP(3)R2, as demonstrated by (32)P labeling. Incorporation of CaMKII-treated InsP(3)R2 into planar lipid bilayers revealed that InsP(3)-mediated channel open probability is significantly reduced ( approximately 11 times) by phosphorylation via CaMKII. We concluded that the InsP(3)R2 and CaMKIIdelta likely represent two central components of a multiprotein signaling complex, and this raises the possibility that calcium release via InsP(3)R2 in the myocyte NE may activate local CaMKII signaling, which may feedback on InsP(3)R2 function.

Inositol 1,4,5-trisphosphate (InsP3) receptors (InsP3Rs) are channels responsible for calcium release from the endoplasmic reticulum (ER). We show that the anti-apoptotic protein Bcl-2 (either wild type or selectively localized to the ER) significantly inhibited InsP3-mediated calcium release and elevation of cytosolic calcium in WEHI7.2 T cells. This inhibition was due to an effect of Bcl-2 at the level of InsP3Rs because responses to both anti-CD3 antibody and a cell-permeant InsP3 ester were decreased. Bcl-2 inhibited the extent of calcium release from the ER of permeabilized WEHI7.2 cells, even at saturating concentrations of InsP3, without decreasing luminal calcium concentration. Furthermore, Bcl-2 reduced the open probability of purified InsP3Rs reconstituted into lipid bilayers. Bcl-2 and InsP3Rs were detected together in macromolecular complexes by coimmunoprecipitation and blue native gel electrophoresis. We suggest that this functional interaction of Bcl-2 with InsP3Rs inhibits InsP3R activation and thereby regulates InsP3-induced calcium release from the ER. ispartof: JOURNAL OF CELL BIOLOGY vol:166 issue:2 pages:193-203 ispartof: location:United States status: published

The three-dimensional structure of the type 1 inositol 1,4,5-trisphosphate receptor (InsP3R1) has been determined by electron cryomicroscopy and single-particle reconstruction. The receptor was immunoaffinity-purified and formed functional InsP3- and heparin-sensitive channels with a unitary conductance similar to native InsP3Rs. The channel structure exhibits the expected 4-fold symmetry and comprises two morphologically distinct regions: a large pinwheel and a smaller square. The pinwheel region has four radial curved spokes interconnected by a central core. The InsP3-binding core domain has been localized within each spoke of the pinwheel region by fitting its x-ray structure into our reconstruction. A structural mapping of the amino acid sequences to several functional domains is deduced within the structure of the InsP3R1 tetramer.

Ryanodine receptor (RyR) phosphorylation by protein kinase A (PKA) may be important in modulating resting sarcoplasmic reticulum (SR) Ca 2+ release, especially in heart failure. However, clear cellular data on PKA-dependent modulation of cardiac RyRs is limited because of difficulty in distinguishing between PKA effects on RyR, phospholamban (PLB), and Ca 2+ current. To clarify this, we measured resting Ca 2+ sparks in streptolysin-O permeabilized ventricular myocytes from wild-type (WT) and PLB knockout (PLB-KO) mice and transgenic mice expressing only double-mutant PLB (PLB-DM) that lacks the regulatory phosphorylation sites (S16A/T17A). In WT myocytes, cAMP dramatically increased Ca 2+ spark frequency (CaSpF) by 2- and 3-fold when [Ca 2+ ] was clamped at 50 and 10 nmol/L (and the SR Ca 2+ content also rose by 40% and 50%). However, in PLB-KO and PLB-DM, neither CaSpF nor SR Ca 2+ load was changed by the addition of 10 μmol/L cAMP (even with phosphatase inhibition). PKA activation also increased Ca 2+ spark amplitude, duration, and width in WT, but not in PLB-KO or PLB-DM. RyR phosphorylation was confirmed by measurements of 32 P incorporation on immunoprecipitated RyR. In intact resting myocytes, PKA activation increased CaSpF 2.8-fold in WT, but not in PLB-KO, confirming results in permeabilized myocytes. We conclude that the PKA-dependent increase in myocyte CaSpF and size is entirely attributable to PLB phosphorylation and consequent enhanced SR Ca 2+ load. PKA does not seem to have any appreciable effect on resting RyR function in these ventricular myocytes. Moreover, the data provide compelling evidence that elevated intra-SR [Ca 2+ ] increases RyR gating independent of cytosolic [Ca 2+ ] (which was clamped).

Potassium depolarization of skeletal myotubes evokes slow calcium waves that are unrelated to contraction and involve the cell nucleus (Jaimovich, E., Reyes, R., Liberona, J. L., and Powell, J. A. (2000) Am. J. Physiol. 278, C998-C1010). Studies were done in both the 1B5 (Ry53-/-) murine "dyspedic" myoblast cell line, which does not express any ryanodine receptor isoforms (Moore, R. A., Nguyen, H., Galceran, J., Pessah, I. N., and Allen, P. D. (1998) J. Cell Biol. 140, 843-851), and C(2)C(12) cells, a myoblast cell line that expresses all three isoforms. Although 1B5 cells lack ryanodine binding, they bind tritiated inositol (1,4,5)-trisphosphate. Both type 1 and type 3 inositol trisphosphate receptors were immuno-located in the nuclei of both cell types and were visualized by Western blot analysis. After stimulation with 47 mm K(+), inositol trisphosphate mass raised transiently in both cell types. Both fast calcium increase and slow propagated calcium signals were seen in C(2)C(12) myotubes. However, 1B5 myotubes (as well as ryanodine-treated C(2)C(12) myotubes) displayed only a long-lasting, non-propagating calcium increase, particularly evident in the nuclei. Calcium signals in 1B5 myotubes were almost completely blocked by inhibitors of the inositol trisphosphate pathway: U73122, 2-aminoethoxydiphenyl borate, or xestospongin C. Results support the hypothesis that inositol trisphosphate mediates slow calcium signals in muscle cell ryanodine receptors, having a role in their time course and propagation.

In the present work, we studied the interaction and effect of several IP3 receptor (IP3R) constructs on the gating of the store-operated (SOC) hTrp3 channel. Full-length IP3R coupled to silent hTrp3 channels in intact cells but did not activate them until stores were depleted of Ca2+. By contrast, constructs containing the IP3-binding domain activated silent hTrp3 channels in unstimulated cells and restored gating of hTrp3 by IP3 in excised plasma membrane patches. We conclude that the N-terminal domain of the IP3R functions as a gate and is sufficient for activation of SOCs. The sensing and transduction domains of the IP3R are required to maintain SOCs in an inactive state.

Calcium ions are released from intracellular stores in response to agonist-stimulated production of inositol 1,4,5-trisphosphate (InsP3), a second messenger generated at the cell membrane. Depletion of Ca2+ from internal stores triggers a capacitative influx of extracellular Ca2+ across the plasma membrane1,2. The influx of Ca2+ can be recorded as store-operated channels (SOC) in the plasma membrane or as a current known as the Ca2+-release-activated current (Icrac)3,4,5. A critical question in cell signalling is how SOC and Icrac sense and respond to Ca2+-store depletion: in one model, a messenger molecule is generated that activates Ca2+ entry in response to store depletion1,6; in an alternative model7, InsP3 receptors in the stores are coupled to SOC and Icrac. The mammalian Htrp3 protein8 forms a well defined store-operated channel8,9 and so provides a suitable system for studying the effect of Ca2+-store depletion on SOC and Icrac. We show here that Htrp3 channels stably expressed in HEK293 cells are in a tight functional interaction with the InsP3 receptors. Htrp3 channels present in the same plasma membrane patch can be activated by Ca2+ mobilization in intact cells and by InsP3 in excised patches. This activation of Htrp3 by InsP3 is lost on extensive washing of excised patches but is restored by addition of native or recombinant InsP3-bound InsP3 receptors. Our results provide evidence for the coupling hypothesis7, in which InsP3 receptors activated by InsP3 interact with SOC and regulate Icrac.

The inositol 1,4,5-trisphosphate receptor (InsP3R) family of Ca2+ release channels is central to intracellular Ca2+ signaling in mammalian cells. The InsP3R channels release Ca2+ from intracellular compartments to generate localized Ca2+ transients that govern a myriad of cellular signaling phenomena (Berridge, 1993. Nature. 361:315–325; Joseph, 1996. Cell Signal. 8:1–7; Kume et al., 1997. Science. 278:1940–1943; Berridge, 1997. Nature. 368:759–760). Most cells express multiple InsP3R isoforms, but only the function of the single type 1 InsP3R channel is known. Here the single-channel function of single type 2 InsP3R channel is defined for the first time. The type 2 InsP3R forms channels with permeation properties similar to that of the type 1 receptor. The InsP3 regulation and Ca2+ regulation of type 1 and type 2 InsP3R channels are strikingly different. Both InsP3 and Ca2+ are more effective at activating single type 2 InsP3R, indicating that single type 2 channels mobilize substantially more Ca2+ than single type 1 channels in cells. Furthermore, high cytoplasmic Ca2+ concentrations inactivate type 1, but not type 2, InsP3R channels. This indicates that type 2 InsP3R channel is different from the type 1 channel in that its activity will not be inherently self-limiting, because Ca2+ passing through an active type 2 channel cannot feed back and turn the channel off. Thus the InsP3R identity will help define the spatial and temporal nature of local Ca2+ signaling events and may contribute to the segregation of parallel InsP3 signaling cascades in mammalian cells.

Publisher Summary This chapter presents the molecular analysis of inositol 1, 4, 5-trisphosphate receptors. Inositol 1, 4, 5-trisphosphate (IP 3 ) is one of the most important second messengers. Production of IP 3 is caused by the activation of receptors for hundreds of biologically active substances. The experiments have shown that IP 3 has a function in a multitude of physiological processes, ranging from muscle contraction to lymphocyte activation and from cell division to neuronal excitation. The IP 3 receptors and the ryanodine receptors are surprisingly large proteins. They are larger than any other current described ligand-gated ion channel. The IP 3 receptor has four currently known functional properties that can be partially investigated using mutagenesis and protein expression: (1) ligand (IP 3 ) binding, (2) tetramerization, (3) IP 3 -dependent Ca 2+ channel gating, and (4) modulation of IP 3 -dependent Ca 2+ channel gating. Functional expression of IP 3 receptors whose Ca 2+ gating could be investigated has not been possible, making it currently unfeasible to investigate the last two properties of IP 3 receptors. The reason for the inability to investigate Ca2+ gating at present time is that all cells contain a very efficient InsP 3 -gated Ca 2+ release pathway. This makes it impossible to use simple Ca 2+ flux measurements in transfected cells. At low concentrations, Ca 2+ is a necessary coagonist for Ca 2+ release by IP 3 , whereas at high concentrations Ca 2+ blocks IP 3 -triggered Ca 2+ release. Therefore, molecular basis of IP 3 is completely regulated by calcium ions.

Type 1 inositol 1,4,5-trisphosphate receptor (IP3R1), a 1.3 MDa tetrameric membrane protein regulates the release of Ca2+ from endoplasmic reticulum stores into the cytoplasm and plays an essential role in a wide variety of cellular processes. We determined the structure of IP3R1 in the closed conformation at ∼9-A resolution by using single particle electron cryomicroscopy. The channel protein was solubilized with detergent from rat cerebellar microsomes and purified by immunoaffinity chromatography. The purified channels were reconstituted into Ca2+-loaded lipid vesicles and released Ca2+ in response to nanomolar concentrations of IP3, indicating their functionality. Isolated IP3R1 particles were embedded in vitreous ice for cryo-EM in the presence of 1 mM EGTA to drive the channel protein into its closed conformation. The 3D density map of IP3R1 was generated with 37,231 particles extracted from 929 CCD frames collected on a JEOL2010F electron cryomicroscope. The reconstruction was performed using EMAN software. This structure allows visualizing a number of alpha-helices in the membrane-spanning region of the IP3R1 channel, including the inner alpha-helices lining the tapering ion conduction pathway. The molecular architecture of the closed pore is established based on the 9-A cryo-EM density map of IP3R1 and via computational and bioinformatics approaches.This research is supported by grants from NIH (R01GM072804, P41RR02250) and MDA Research Grant 88677.

We reported that inhibition of adenylate cyclase (AC)/cAMP by cell attachment to laminin (+LMN) or pharmacological (KT5720) inhibition of cAMP-dependent protein kinase (PKA) in cells not attached to LMN (-LMN-PKA), activates β2-AR stimulation of ICa,L via cPLA2 signaling. The present study determined the role of PKC and IP3-mediated Ca2+ release in β2-AR/cPLA2 signaling. As previously reported, 0.1 μM zinterol (β2-AR agonist) stimulation of L-type Ca2+ current (ICa,L) was unaffected by 10 μM AACOCF3 (cPLA2 inhibitor) in cells not attached to LMN (-LMN) but was significantly inhibited in +LMN and -LMN-PKA myocytes. Zinterol stimulation of ICa,L in -LMN-PKA myocytes was blocked by 5 μM U73122 (PLC inhibitor) and significantly inhibited by 4 μM chelerythrine (PKC inhibitor). Zinterol stimulation of ICa,L in -LMN myocytes was unaffected by inhibition of IP3-receptors (IP3Rs) by 2 μM 2-APB, but was significantly inhibited in +LMN and -LMN-PKA myocytes. Cells were cultured on LMN (24 hrs) with an adenovirus IP3 affinity trap to inhibit IP3-dependent Ca2+ signaling. Compared to control cells (β-gal), zinterol stimulation of ICa,L was significantly inhibited in cells infected with IP3 trap. Laser scanning confocal microscopy (fluo-4) revealed that zinterol stimulation of +LMN myocytes elicited local intracellular Ca2+ release events in 1 mM tetracaine (blocks RyR Ca2+ release), that were blocked by 2-APB. We conclude that inhibition of cAMP/PKA activates β2-AR stimulation of ICa,L via cPLA2 which is dependent on PKC and IP3-mediated Ca2+ signaling. These findings may be relevant to the remodeling of β-AR signaling in diseased (fibrotic) and/or aging atria, both of which exhibit decreases in AC activity.

The nature of second messenger-responsive intracellular Ca2+ stores in neurons remains open for discussion. Here, we demonstrate the existence in Purkinje cells (PCs) of endoplastic reticulum (ER) subcompartments characterized by an uneven distribution of three proteins involved in Ca2+ storage and release: the inositol 1,4,5-trisphosphate (InsP3) receptor, Ca(2+)-ATPase, and calsequestrin. Ca(2+)-ATPase and the InsP3 receptor have a widespread, although not identical, distribution throughout the ER. Calsequestrin is localized throughout the smooth ER and is particularly concentrated in pleiomorphic vesicles with a moderately electron-dense core, which appear to represent a subcompartment of the smooth ER. In double-labeling experiments many of these vesicles were unlabeled by InsP3 receptor antibodies. These results suggest a key role of the ER as an intracellular Ca2+ store and demonstrate a possible structural basis for distinct intracellular Ca2+ pools regulated by different second messengers.

InsP3-mediated intracellular Ca transients can activate Ca2+-calmodulin-dependent protein kinase II (CaMKII), a multifunctional Serine/Threonine protein kinase involved in many signaling pathways. Recent results show that InsP3Rs in the heart (InsP3R2) are primarily targeted to the nuclear envelope in ventricular cardiac myocytes. Here it forms a macromolecular complex with CaMKIIδ (Bare et al, 2005, JBC) . Upon stimulation of InsP3 production, Ca2+ released through the InsP3R2 activates CaMKIIδ, allowing it to act on downstream targets, such as histone deacetylases 4 & 5 (HDAC4 & HDAC5) (Zhang et al, 2007, JBC). Additionally, CaMKII activity feedback modulates InsP3R2 function by direct phosphorylation and results in a decrease in the channel's open probability. The results of this study show that in planar lipid bilayers the channel activity of InsP3Rs can be inhibited by CaMKII-mediated phosphorylation, and that effect can be reversed by addition of protein phosphatases. Furthermore, the N-terminal 1078 amino acids of the rat InsP3R2 have been shown to interact with, as well as be phosphorylated by CaMKII in in vitro kinase assays. A smaller fragment spanning amino acids 1-708 of the InsP3R2 has been shown to be phosphorylated in a CaMKII-dependent manner. Conversely, C-terminal regions were not phosphorylated by CaMKII in vitro. We have also shown that the N-terminal region of the rat InsP3R1 spanning amino acids 1-1081 can be phosphorylated by CaMKII. Our results from mass spectrometry and in vitro kinase assays indicate that the putative CaMKII regulatory phosphorylation site lies within amino acids 150-340 of the InsP3R2.This work was supported by National Institutes of Health Grant HL-80101