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The Orai1 channel is regulated by stromal interaction molecules STIM1 and STIM2 within endoplasmic reticulum (ER)-plasma membrane (PM) contact sites. Ca 2+ signals generated by Orai1 activate Ca 2+ -dependent gene expression. When compared with STIM1, STIM2 is a weak activator of Orai1, but it has been suggested to have a unique role in nuclear factor of activated T cells 1 (NFAT1) activation triggered by Orai1-mediated Ca 2+ entry. In this study, we examined the contribution of STIM2 in NFAT1 activation. We report that STIM2 recruitment of Orai1/STIM1 to ER-PM junctions in response to depletion of ER-Ca 2+ promotes assembly of the channel with AKAP79 to form a signaling complex that couples Orai1 channel function to the activation of NFAT1. Knockdown of STIM2 expression had relatively little effect on Orai1/STIM1 clustering or local and global [Ca 2+ ] i increases but significantly attenuated NFAT1 activation and assembly of Orai1 with AKAP79. STIM1ΔK, which lacks the PIP 2 -binding polybasic domain, was recruited to ER-PM junctions following ER-Ca 2+ depletion by binding to Orai1 and caused local and global [Ca 2+ ] i increases comparable to those induced by STIM1 activation of Orai1. However, in contrast to STIM1, STIM1ΔK induced less NFAT1 activation and attenuated the association of Orai1 with STIM2 and AKAP79. Orai1-AKAP79 interaction and NFAT1 activation were recovered by coexpressing STIM2 with STIM1ΔK. Replacing the PIP 2 -binding domain of STIM1 with that of STIM2 eliminated the requirement of STIM2 for NFAT1 activation. Together, these data demonstrate an important role for STIM2 in coupling Orai1-mediated Ca 2+ influx to NFAT1 activation., Competing Interests: Competing interest statement: S.F. is a scientific cofounder of CalciMedica.

The intracellular calcium signaling processes are tightly regulated to ensure the generation of calcium signals with the specific spatiotemporal characteristics required for regulating various cell functions. Compartmentalization of the molecular components involved in the generation of these signals at discrete intracellular sites ensures the signaling specificity and transduction fidelity of the signal for regulating downstream effector processes. Store-operated calcium entry (SOCE) is ubiquitously present in cells and is critical for essential cell functions in a variety of tissues. SOCE is mediated via plasma membrane Ca 2+ channels that are activated when luminal [Ca 2+ ] of the endoplasmic reticulum ([Ca 2+ ] ER ) is decreased. The ER-resident stromal interaction molecules, STIM1 and STIM2, respond to decreases in [Ca 2+ ] ER by undergoing conformational changes that cause them to aggregate at the cell periphery in ER-plasma membrane (ER-PM) junctions. At these sites, STIM proteins recruit Orai1 channels and trigger their activation. Importantly, the two STIM proteins concertedly modulate Orai1 function as well as the sensitivity of SOCE to ER-Ca 2+ store depletion. Another family of plasma membrane Ca 2+ channels, known as the Transient Receptor Potential Canonical (TRPC) channels (TRPC1-7) also contribute to sustained [Ca 2+ ] i elevation. Although Ca 2+ signals generated by these channels overlap with those of Orai1, they regulate distinct functions in the cells. Importantly, STIM1 is also required for plasma membrane localization and activation of some TRPCs. In this review, we will discuss various molecular components and factors that govern the activation, regulation and modulation of the Ca 2+ signal generated by Ca 2+ entry pathways in response to depletion of ER-Ca 2+ stores. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech., (Published by Elsevier B.V.)

Store-operated Ca 2+ entry (SOCE) is a ubiquitous pathway for Ca 2+ influx across the plasma membrane (PM). SOCE is mediated by the endoplasmic reticulum (ER)-associated Ca 2+ -sensing proteins stromal interaction molecule 1 (STIM1) and STIM2, which transition into an active conformation in response to ER Ca 2+ store depletion, thereby interacting with and gating PM-associated ORAI1 channels. Although structurally homologous, STIM1 and STIM2 generate distinct Ca 2+ signatures in response to varying strengths of agonist stimulation. The physiological functions of these Ca 2+ 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 Ca 2+ 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.

Store-operated calcium entry (SOCE), a unique plasma membrane Ca 2+ entry mechanism, is activated when ER-[Ca 2+ ] is decreased. SOCE is mediated via the primary channel, Orai1, as well as others such as TRPC1. STIM1 and STIM2 are ER-Ca 2+ 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 Ca 2+ 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 Ca 2+ signals that are critical for regulating many downstream cellular functions.

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., (© 2016 The Authors. The Journal of Physiology © 2016 The Physiological Society.)

Background/aims: Adenosine diphosphate ribose (ADPR), a product of β-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., (© 2015 S. Karger AG, Basel.)

Voltage-dependent anion channels (VDACs) are pore forming proteins predominantly found in the outer mitochondrial membrane and are thought to transport Ca(2+). In this study, we have investigated the possible role of type 2 VDAC (VDAC2) in cardiac Ca(2+) signaling and Ca(2+) sparks using a lentiviral knock-down (KD) technique and two-dimensional confocal Ca(2+) imaging in immortalized autorhythmic adult atrial cells, HL-1. We confirmed high expression of VDAC2 protein in ventricular, atrial, and HL-1 cells using Western blot analysis. Infection of HL-1 cells with VDAC2-targeting lentivirus reduced the level of VDAC2 protein to ∼10%. Comparisons of autorhythmic Ca(2+) transients between wild-type (WT) and VDAC2 KD cells showed no significant change in the magnitude, decay, and beating rate of the Ca(2+) transients. Caffeine (10mM)-induced Ca(2+) release, which indicates sarcoplasmic reticulum (SR) Ca(2+) content, was not altered by VDAC2 KD. Interestingly, however, the intensity, width, and duration of the individual Ca(2+) sparks were significantly increased by VDAC2 KD in resting conditions, with no change in the frequency of sparks. VDAC2 KD significantly delayed mitochondrial Ca(2+) uptake during artificial Ca(2+) pulses in permeabilized HL-1 cells. These results suggest that VDAC2 may facilitate mitochondrial Ca(2+) uptake and restrict Ca(2+) spark expansion without regulating activations of sparks under resting conditions, thereby providing evidence on the functional role of VDAC2 in cardiac local Ca(2+) signaling., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

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., (2010 Elsevier Ltd. All rights reserved.)