Cross-talk between N-terminal and C-terminal domains in stromal interaction molecule 2 (STIM2) determines enhanced STIM2 sensitivity.

Store-operated Ca2+ entry (SOCE) is a ubiquitous pathway for Ca2+ influx across the plasma membrane (PM). SOCE is mediated by the endoplasmic reticulum (ER)-associated Ca2+-sensing proteins stromal interaction molecule 1 (STIM1) and STIM2, which transition into an active conformation in response to ER Ca2+ store depletion, thereby interacting with and gating PM-associated ORAI1 channels. Although structurally homologous, STIM1 and STIM2 generate distinct Ca2+ signatures in response to varying strengths of agonist stimulation. The physiological functions of these Ca2+ signatures, particularly under native conditions, remain unclear. To investigate the structural properties distinguishing STIM1 and STIM2 activation of ORAI1 channels under native conditions, here we used CRISPR/Cas9 to generate STIM1-/-, STIM2-/-, and STIM1/2-/- knockouts in HEK293 and colorectal HCT116 cells. We show that depending on cell type, STIM2 can significantly sustain SOCE in response to maximal store depletion. Utilizing the SOCE modifier 2-aminoethoxydiphenyl borate (2-APB), we demonstrate that 2-APB-activated store-independent Ca2+ entry is mediated exclusively by endogenous STIM2. Using variants that either stabilize or disrupt intramolecular interactions of STIM C termini, we show that the increased flexibility of the STIM2 C terminus contributes to its selective store-independent activation by 2-APB. However, STIM1 variants with enhanced flexibility in the C terminus failed to support its store-independent activation. STIM1/STIM2 chimeric constructs indicated that coordination between N-terminal sensitivity and C-terminal flexibility is required for specific store-independent STIM2 activation. Our results clarify the structural determinants underlying activation of specific STIM isoforms, insights that are potentially useful for isoform-selective drug targeting.

Signaling Pathway for Endothelin-1- and Phenylephrine-Induced cAMP Response Element Binding Protein Activation in Rat Ventricular Myocytes: Role of Inositol 1,4,5-Trisphosphate Receptors and CaMKII.

BACKGROUND/AIMS: Endothelin-1 (ET-1) and the α₁-adrenoceptor agonist phenylephrine (PE) activate cAMP response element binding protein (CREB), a transcription factor implicated in cardiac hypertrophy. The signaling pathway involved in CREB activation by these hypertrophic stimuli is poorly understood. We examined signaling pathways for ET-1- or PE-induced cardiac CREB activation. METHODS: Western blotting was performed with pharmacological and genetic interventions in rat ventricular myocytes. RESULTS: ET-1 and PE increased CREB phosphorylation, which was inhibited by blockade of phospholipase C, the extracellular-signal-regulated kinase 1/2 (ERK1/2) pathway, protein kinase C (PKC) or Ca2+-calmodulin-dependent protein kinase II (CaMKII). Intracellular Ca2+ buffering decreased ET-1- and PE-induced CREB phosphorylation by ≥80%. Sarcoplasmic reticulum Ca2+ pump inhibitor, inositol 1,4,5-trisphosphate receptor (IP₃R) blockers, or type 2 IP₃R (IP₃R2) knock-out abolished ET-1- or PE-induced CREB phosphorylation. ET-1 and PE increased phosphorylation of CaMKII and ERK1/2, which was eliminated by IP₃R blockade/knock-out or PKC inhibition. Activation of CaMKII, but not ERK1/2, by these agonists was sensitive to Ca2+ buffering or to Gö6976, the inhibitor of Ca2+-dependent PKC and protein kinase D (PKD). CONCLUSION: CREB phosphorylation by ET-1 and PE may be mainly mediated by IP₃R2/Ca2+-PKC-PKD-CaMKII signaling with a minor contribution by ERK1/2, linked to IP₃R2 and Ca2+-independent PKC, in ventricular myocytes.

Assembly of ER-PM Junctions: A Critical Determinant in the Regulation of SOCE and TRPC1.

Store-operated calcium entry (SOCE), a unique plasma membrane Ca2+ entry mechanism, is activated when ER-[Ca2+] is decreased. SOCE is mediated via the primary channel, Orai1, as well as others such as TRPC1. STIM1 and STIM2 are ER-Ca2+ sensor proteins that regulate Orai1 and TRPC1. SOCE requires assembly of STIM proteins with the plasma membrane channels which occurs within distinct regions in the cell that have been termed as endoplasmic reticulum (ER)-plasma membrane (PM) junctions. The PM and ER are in close proximity to each other within this region, which allows STIM1 in the ER to interact with and activate either Orai1 or TRPC1 in the plasma membrane. Activation and regulation of SOCE involves dynamic assembly of various components that are involved in mediating Ca2+ entry as well as those that determine the formation and stabilization of the junctions. These components include proteins in the cytosol, ER and PM, as well as lipids in the PM. Recent studies have also suggested that SOCE and its components are compartmentalized within ER-PM junctions and that this process might require remodeling of the plasma membrane lipids and reorganization of structural and scaffolding proteins. Such compartmentalization leads to the generation of spatially- and temporally-controlled Ca2+signals that are critical for regulating many downstream cellular functions.

Shear stress activates monovalent cation channel transient receptor potential melastatin subfamily 4 in rat atrial myocytes via type 2 inositol 1,4,5-trisphosphate receptors and Ca(2+) release.

KEY POINTS: During each contraction and haemodynamic disturbance, cardiac myocytes are subjected to fluid shear stress as a result of blood flow and the relative movement of sheets of myocytes. The present study aimed to characterize the shear stress-sensitive membrane current in atrial myocytes using the whole-cell patch clamp technique, combined with pressurized fluid flow, as well as pharmacological and genetic interventions of specific proteins. The data obtained suggest that shear stress indirectly activates the monovalent cation current carried by transient receptor potential melastatin subfamily 4 channels via type 2 inositol 1,4,5-trisphosphate receptor-mediated Ca(2+) release in subsarcolemmal domains of atrial myocytes. Ca(2+) -mediated interactions between these two proteins under shear stress may be an important mechanism by which atrial cells measure mechanical stress and translate it to alter their excitability. ABSTRACT: Atrial myocytes are subjected to shear stress during the cardiac cycle under physiological or pathological conditions. The ionic currents regulated by shear stress remain poorly understood. We report the characteristics, molecular identity and activation mechanism of the shear stress-sensitive current (Ishear ) in rat atrial myocytes. A shear stress of ∼16 dyn cm(-2) was applied to single myocytes using a pressurized microflow system, and the current was measured by whole-cell patch clamp. In symmetrical CsCl solutions with minimal concentrations of internal EGTA, Ishear showed an outwardly rectifying current-voltage relationship (reversal at -2 mV). The current was conducted primarily (∼80%) by monovalent cations but not Ca(2+) . It was suppressed by intracellular Ca(2+) buffering at a fixed physiological level, inhibitors of transient receptor potential melastatin subfamily 4 (TRPM4), intracellular introduction of TRPM4 antibodies or knockdown of TRPM4 expression, suggesting that TRPM4 carries most of this current. A notable reduction in Ishear occurred upon inhibition of Ca(2+) release through the ryanodine receptors or inositol 1,4,5-trisphosphate receptors (IP3 R) and upon depletion of sarcoplasmic reticulum Ca(2+) . In type 2 IP3 R (IP3 R2) knockout atrial myocytes, Ishear was 10-20% of that in wild-type myocytes. Immunocytochemistry and proximity ligation assays revealed that TRPM4 and IP3 R2 were expressed at peripheral sites with co-localization, although they are not localized within 40 nm. Peripheral localization of TRPM4 was intact in IP3 R2 knockout cells. The data obtained in the present study suggest that shear stress activates TRPM4 current by triggering Ca(2+) release from the IP3 R2 in the peripheral domains of atrial myocytes.

Extracellular Adenosine Diphosphate Ribose Mobilizes Intracellular Ca2+ via Purinergic-Dependent Ca2+ Pathways in Rat Pulmonary Artery Smooth Muscle Cells.

BACKGROUND/AIMS: Adenosine diphosphate ribose (ADPR), a product of ß-NAD+ metabolism generated by the multifunctional enzyme CD38, is recognized as a novel signaling molecule. The catalytic site of CD38 orients extracellularly or intracellularly, capable of generating ADPR outside and inside the cells. CD38-dependent pathways have been characterized in pulmonary artery smooth muscle cells (PASMCs); however the physiological function of extracellular ADPR is unclear. METHODS: Ca2+ mobilizing and proliferative effects of extracellular ADPR were characterized and compared with the ATP-induced responses in rat PASMCs; and the expression of purinergic receptor (P2X and P2Y) subtypes were examined in pulmonary arteries. RESULTS: ADPR elicited concentration-dependent increase in [Ca2+]i with a fast transient and a sustained phase in PASMCs. The sustained phase was abolished by Ca2+ removal and inhibited by the non-selective cation channel blocker SKF-96365, but was unaffected by TRPM2 antagonists or nifedipine. The purinergic receptor (P2X) antagonist pyridoxal-phosphate-6-azophenyl-2', 4'-disulfonate inhibited partially the transient and the sustained Ca2+ response, while the P2(XY) inhibitor suramin and the phospholipase C inhibitor U73122 abolished the sustained Ca2+ influx. The P2Y1 antagonist MRS2179 had no effect on the response. By contrast, ATP and ADP activated Ca2+ response exhibited a high and a low affinity component, and the pharmacological profile of ATP-induced Ca2+ response was distinctive from that of ADPR. BrdU incorporation assay showed that ADPR caused significant inhibition whereas ATP caused slight stimulation of PASMC proliferation. RT-PCR analysis found that almost all P2X and P2Y subtypes are expressed in PAs. CONCLUSION: ADPR and ATP activate Ca2+ responses through different combinations of multiple purinergic receptor subtypes; and extracellular ADPR may exert an autocrine/paracrine action via purinergic receptors on PASMCs.

Detection of differentially regulated subsarcolemmal calcium signals activated by vasoactive agonists in rat pulmonary artery smooth muscle cells.

Intracellular calcium (Ca(2+)) plays pivotal roles in distinct cellular functions through global and local signaling in various subcellular compartments, and subcellular Ca(2+) signal is the key factor for independent regulation of different cellular functions. In vascular smooth muscle cells, subsarcolemmal Ca(2+) is an important regulator of excitation-contraction coupling, and nucleoplasmic Ca(2+) is crucial for excitation-transcription coupling. However, information on Ca(2+) signals in these subcellular compartments is limited. To study the regulation of the subcellular Ca(2+) signals, genetically encoded Ca(2+) indicators (cameleon), D3cpv, targeting the plasma membrane (PM), cytoplasm, and nucleoplasm were transfected into rat pulmonary arterial smooth muscle cells (PASMCs) and Ca(2+) signals were monitored using laser scanning confocal microscopy. In situ calibration showed that the Kd for Ca(2+) of D3cpv was comparable in the cytoplasm and nucleoplasm, but it was slightly higher in the PM. Stimulation of digitonin-permeabilized cells with 1,4,5-trisphosphate (IP3) elicited a transient elevation of Ca(2+) concentration with similar amplitude and kinetics in the nucleoplasm and cytoplasm. Activation of G protein-coupled receptors by endothelin-1 and angiotensin II preferentially elevated the subsarcolemmal Ca(2+) signal with higher amplitude in the PM region than the nucleoplasm and cytoplasm. In contrast, the receptor tyrosine kinase activator, platelet-derived growth factor, elicited Ca(2+) signals with similar amplitudes in all three regions, except that the rise-time and decay-time were slightly slower in the PM region. These data clearly revealed compartmentalization of Ca(2+) signals in the subsarcolemmal regions and provide the basis for further investigations of differential regulation of subcellular Ca(2+) signals in PASMCs.

Hsp31 of Escherichia coli K-12 is glyoxalase III.

Hsp31 encoded by hchA is known as a heat-inducible molecular chaperone. Although structure studies revealed that Hsp31 has a putative catalytic triad consisting of Asp-214, His-186 and Cys-185, its enzymatic function, besides weak amino-peptidase activity, is still unknown. We found that Hsp31 displays glyoxalase activity that catalyses the conversion of methylglyoxal (MG) to d-lactate without an additional cofactor. The glyoxalase activity was completely abolished in the hchA-deficient strain, confirming the relationship between the hchA gene and its enzymatic activity in vivo. Hsp31 exhibits Michaelis-Menten kinetics for substrates MG with K(m) and k(cat) of 1.43±0.12 mM and 156.9±5.5 min⁻¹ respectively. The highest glyoxalase activity was found at 35-40 °C and pH of 6.0-8.0, and the activity was significantly inhibited by Cu²âº, Fe³âº and Zn²âº. Mutagenesis studies based on our evaluation of conserved catalytic residues revealed that the Cys-185 and Glu-77 were essential for catalysis, whereas His-186 was less crucial for enzymatic function, although it participates in the catalytic process. The stationary-phase Escherichia coli cells became more susceptible to MG when hchA was deleted, which was complemented by an expression of plasmid-encoded hchA. Furthermore, an accumulation of intracellular MG was observed in hchA-deficient strains.

Cloning and expression of a new inositol 1,4,5-trisphosphate receptor type 1 splice variant in adult rat atrial myocytes.

Inositol 1,4,5-trisphosphate receptor type 1 (IP(3)R1) is already known to be highly expressed in the brain, and is found in many other tissues, including the atrium of the heart. Although the complete primary structure of IP(3)R1 in the rat brain has been reported, the complete sequence of an IP(3)R1 clone from atrial myocytes has not been reported. We isolated an IP(3)R1 complementary DNA (cDNA) clone from isolated adult rat atrial myocytes, and found a new splice variant of IP(3)R1 that was different from a previously reported IP(3)R1 cDNA clone obtained from a rat brain (NCBI GenBank accession number: NM_001007235). Our clone had 99% similarity with the rat brain IP(3)R1 sequence; the exceptions were 39 amino acid deletions at the position of 1693-1731, and the deletion of phenylalanine at position 1372 that lay in the regulatory region. Compared with the rat brain IP(3)R1, in our clone proline was replaced with serine at residue 2439, and alanine was substituted for valine at residue 2445. These changes lie adjacent to or within the fifth transmembrane domain (2440-2462). Although such changes in the amino acid sequences were different from the rat brain IP3R1 clone, they were conserved in human or mouse IP3R1. We produced a plasmid construct expressing the atrial IP3R1 together with green fluorescent protein (GFP), and successfully overexpressed the atrial IP3R1 in the adult atrial cell line HL-1. Further investigation is needed on the physiological significance of the new splice variant in atrial cell function.

Jejunojejunal intussusception in an adult due to adenoma: a case report.

Intussusception is a rare condition in adults and presents a diagnostic challenge. Clinical presentation tends to be chronic and non-specific. Unlike the pediatric population, most adult intussusceptions have structural lesions as lead points. Here, we present a case of jejunojejunal intussusception in a 27-year female due to adenoma of small bowel.

Injuries and deaths due to victim-activated improvised explosive devices, landmines and other explosive remnants of war in Nepal.

BACKGROUND: Following more than a decade of civil conflict, Nepal is among the countries affected by landmines, victim-activated improvised explosive devices (IED) and other explosive remnants of war (ERW). OBJECTIVES: To assess the magnitude of injuries due to landmines, victim-activated IED and other ERW in Nepal and to describe epidemiological patterns and risk factors for these events. METHODS: Analysis of surveillance data on civilian injuries due to landmines, victim-activated IED and other ERW between July 2006 and June 2010. Data were collected through active community-based prospective surveillance. RESULTS: Of 307 total casualties, 94 (31%) were female and 169 (55%) were children under 18 years of age. The case-fatality ratio was 14%. The highest number of casualties was in the age group 10-14 years. 233 (76%) injuries were caused by victim-activated IED, 13 (4%) by landmines and 44 (14%) by other ERW. Two types of IED, sutali and socket bombs, caused the majority of injuries (28% and 31%, respectively). 117 (38%) of all injuries occurred in victims' homes and 152 (50%) occurred while victims were tampering with explosive devices. CONCLUSIONS: Substantial numbers of civilians, including women and children, were injured and killed following implementation of the Comprehensive Peace Agreement in 2006. The government of Nepal and humanitarian organisations should continue their efforts to reach communities at highest risk through targeted interventions and nationwide media campaigns to convey the risks of tampering with explosive devices or suspicious objects.

Mitochondria-targeted antioxidant protects against irradiation-induced salivary gland hypofunction.

A severe consequence of radiation therapy in patients with head and neck cancer is persistent salivary gland hypofunction which causes xerostomia and oral infections. We previously showed that irradiation (IR) of salivary glands in mice triggers initial transient increases in mitochondrial reactive oxygen species (ROSmt), mitochondrial [Ca2+] ([Ca2+]mt), and activated caspase-3 in acinar cells. In contrast, loss of salivary secretion is persistent. Herein we assessed the role of ROSmt in radiation-induced irreversible loss of salivary gland function. We report that treatment of mice with the mitochondrial-targeted antioxidant, MitoTEMPO, resulted in almost complete protection of salivary gland secretion following either single (15 Gy) or fractionated (5 × 3 Gy) doses of irradiation. Salivary gland cells isolated from MitoTEMPO-treated, irradiated, mice displayed significant attenuation of the initial increases in ROSmt, ([Ca2+]mt, and activated caspase-3 as compared to cells from irradiated, but untreated, animals. Importantly, MitoTEMPO treatment prevented radiation-induced decrease in STIM1, consequently protecting store-operated Ca2+ entry which is critical for saliva secretion. Together, these findings identify the initial increase in ROSmt, that is induced by irradiation, as a critical driver of persistent salivary gland hypofunction. We suggest that the mitochondrially targeted antioxidant, MitoTEMPO, can be potentially important in preventing IR-induced salivary gland dysfunction.

Role of GldA in dihydroxyacetone and methylglyoxal metabolism of Escherichia coli K12.

The metabolic pathway involving dihydroxyacetone is poorly characterized although novel enzymes associated with this metabolite have recently been demonstrated. The role of GldA in dihydroxyacetone and methylglyoxal metabolism was investigated by purifying the enzyme and characterizing its catalytic ability using nuclear magnetic resonance (NMR) spectroscopy. At neutral pH, the enzyme exhibits much higher affinities towards dihydroxyacetone, methylglyoxal, and glycolaldehyde than glycerol with K(m) values of 0.30, 0.50, 0.85, and 56 mM, respectively. This is consistent with NMR data with crude extracts, showing that the conversion from dihydroxyacetone to glycerol by GldA is far more efficient than the reverse reaction. Dihydroxyacetone was found to be lethal at higher concentration with an LC(50) value of 28 mM compared with 0.4 mM of methylglyoxal, while lactaldehyde was found to exhibit significant growth inhibition in Escherichia coli cells. The toxicity of dihydroxyacetone appears to be due to its intracellular conversion to an aldehyde compound, presumably methylglyoxal, since the glyoxalase mutant becomes sensitive to dihydroxyacetone. Based on information that gldA is preceded in an operon by the ptsA homolog and talC gene encoding fructose 6-phosphate aldolase, this study proposes that the primary role of gldA is to remove toxic dihydroxyacetone by converting it into glycerol.

Radiation inhibits salivary gland function by promoting STIM1 cleavage by caspase-3 and loss of SOCE through a TRPM2-dependent pathway.

Store-operated Ca2+ entry (SOCE) is critical for salivary gland fluid secretion. We report that radiation treatment caused persistent salivary gland dysfunction by activating a TRPM2-dependent mitochondrial pathway, leading to caspase-3-mediated cleavage of stromal interaction molecule 1 (STIM1) and loss of SOCE. After irradiation, acinar cells from the submandibular glands of TRPM2+/+ , but not those from TRPM2-/- mice, displayed an increase in the concentrations of mitochondrial Ca2+ and reactive oxygen species, a decrease in mitochondrial membrane potential, and activation of caspase-3, which was associated with a sustained decrease in STIM1 abundance and attenuation of SOCE. In a salivary gland cell line, silencing the mitochondrial Ca2+ uniporter or caspase-3 or treatment with inhibitors of TRPM2 or caspase-3 prevented irradiation-induced loss of STIM1 and SOCE. Expression of exogenous STIM1 in the salivary glands of irradiated mice increased SOCE and fluid secretion. We suggest that targeting the mechanisms underlying the loss of STIM1 would be a potentially useful approach for preserving salivary gland function after radiation therapy.

Fatal and non-fatal injuries due to intentional explosions in Nepal, 2008-2011: analysis of surveillance data.

BACKGROUND: Nepal is one of the post-conflict countries affected by violence from explosive devices. We undertook this study to assess the magnitude of injuries due to intentional explosions in Nepal during 2008-2011 and to describe time trends and epidemiologic patterns for these events. METHODS: We analyzed surveillance data on fatal and non-fatal injuries due to intentional explosions in Nepal that occurred between 1 January 2008 and 31 December 2011. The case definition included casualties injured or killed by explosive devices knowingly activated by an individual or a group of individuals with the intent to harm, hurt or terrorize. Data were collected through media-based and active community-based surveillance. RESULTS: Analysis included 437 casualties injured or killed in 131 intentional explosion incidents. A decrease in the number of incidents and casualties between January 2008 and June 2009 was followed by a pronounced increase between July 2010 and June 2011. Eighty-four (19.2%) casualties were among females and 40 (9.2%) were among children under 18 years of age. Fifty-nine (45.3%) incidents involved one casualty, 47 (35.9%) involved 2 to 4 casualties, and 6 involved more than 10 casualties. The overall case-fatality ratio was 7.8%. The highest numbers of incidents occurred in streets or at crossroads, in victims' homes, and in shops or markets. Incidents on buses and near stadiums claimed the highest numbers of casualties per incident. Socket, sutali, and pressure cooker bombs caused the highest numbers of incidents. CONCLUSIONS: Intentional explosion incidents still pose a threat to the civilian population of Nepal. Most incidents are caused by small homemade explosive devices and occur in public places, and males aged 20 to 39 account for a plurality of casualties. Stakeholders addressing the explosive device problem in Nepal should continue to use surveillance data to plan interventions.

IP3-induced cytosolic and nuclear Ca2+ signals in HL-1 atrial myocytes: possible role of IP3 receptor subtypes.

HL-1 cells are the adult cardiac cell lines available that continuously divide while maintaining an atrial phenotype. Here we examined the expression and localization of inositol 1,4,5-trisphosphate receptor (IP(3)R) subtypes, and investigated how pattern of IP(3)-induced subcellular local Ca(2+) signaling is encoded by multiple IP(3)R subtypes in HL-1 cells. The type 1 IP(3)R (IP(3)R1) was expressed in the perinucleus with a diffuse pattern and the type 2 IP(3)R (IP(3)R2) was expressed in the cytosol with a punctate distribution. Extracellular ATP (1 mM) elicited transient intracellular Ca(2+) releases accompanied by a Ca(2+) oscillation, which was eliminated by the blocker of IP(3)Rs, 2-APB, and attenuated by ryanodine. Direct introduction of IP(3) into the permeabilized cells induced Ca(2+) transients with Ca(2+) oscillations at [Symbol: see text] 20 muM of IP(3), which was removed by the inhibition of IP(3)Rs using 2-APB and heparin. IP(3)-induced local Ca(2+) transients contained two distinct time courses: a rapid oscillation and a monophasic Ca(2+) transient. The magnitude of Ca(2+) oscillation was significantly larger in the cytosol than in the nucleus, while the monophasic Ca(2+) transient was more pronounced in the nucleus. These results provide evidence for the molecular and functional expression of IP(3)R1 and IP(3)R2 in HL-1 cells, and suggest that such distinct local Ca(2+) signaling may be correlated with the punctate distribution of IP(3)R2s in the cytosol and the diffuse localization of IP(3)R1 in the peri-nucleus.

Atrial local Ca2+ signaling and inositol 1,4,5-trisphosphate receptors.

In atrial myocytes lacking t-tubules, action potential triggers junctional Ca(2+) releases in the cell periphery, which propagates into the cell interior. The present article describes growing evidence on atrial local Ca(2+) signaling and on the functions of inositol 1,4,5-trisphosphate receptors (IP(3)Rs) in atrial myocytes, and show our new findings on the role of IP(3)R subtype in the regulation of spontaneous focal Ca(2+) releases in the compartmentalized areas of atrial myocytes. The Ca(2+) sparks, representing focal Ca(2+) releases from the sarcoplasmic reticulum (SR) through the ryanodine receptor (RyR) clusters, occur most frequently at the peripheral junctions in isolated resting atrial cells. The Ca(2+) sparks that were darker and longer lasting than peripheral and non-junctional (central) sparks, were found at peri-nuclear sites in rat atrial myocytes. Peri-nuclear sparks occurred more frequently than central sparks. Atrial cells express larger amounts of IP(3)Rs compared with ventricular cells and possess significant levels of type 1 IP(3)R (IP(3)R1) and type 2 IP(3)R (IP(3)R2). Over the last decade the roles of atrial IP(3)R on the enhancement of Ca(2+)-induced Ca(2+) release and arrhythmic Ca(2+) releases under hormonal stimulations have been well documented. Using protein knock-down method and confocal Ca(2+) imaging in conjunction with immunocytochemistry in the adult atrial cell line HL-1, we could demonstrate a role of IP(3)R1 in the maintenance of peri-nuclear and non-junctional Ca(2+) sparks via stimulating a posttranslational organization of RyR clusters.