Activation Of Store-operated Channels Research Articles

A Novel Modulator of STIM2-Dependent Store-Operated Ca2+ Channel Activity.

Store-operated Ca2+ entry is one of the main pathways of calcium influx into non-excitable cells, which entails the initiation of many intracellular processes. The endoplasmic reticulum Ca2+ sensors STIM1 and STIM2 are the key components of store-operated Ca2+ entry in mammalian cells. Under physiological conditions, STIM proteins are responsible for store-operated Ca2+ entry activation. The STIM1 and STIM2 proteins differ in their potency for activating different store-operated channels. At the moment, there are no selective modulators of the STIM protein activity. We screened a library of small molecules and found the 4-MPTC compound, which selectively inhibited STIM2-dependent store-operated Ca2+ entry (IC50 = 1 μM) and had almost no effect on the STIM1-dependent activation of store-operated channels.

STIM1 and STIM2 Proteins Differently Regulate Endogenous Store-operated Channels in HEK293 Cells

The endoplasmic reticulum calcium sensors stromal interaction molecules 1 and 2 (STIM1 and STIM2) are key modulators of store-operated calcium entry. Both these sensors play a major role in physiological functions in normal tissue and in pathology, but available data on native STIM2-regulated plasma membrane channels are scarce. Only a few studies have recorded STIM2-induced CRAC (calcium release-activated calcium) currents. On the other hand, many cell types display store-operated currents different from CRAC. The STIM1 protein regulates not only CRAC but also transient receptor potential canonical (TRPC) channels, but it has remained unclear whether STIM2 is capable of regulating store-operated non-CRAC channels. Here we present for the first time experimental evidence for the existence of endogenous non-CRAC STIM2-regulated channels. As shown in single-channel patch clamp experiments on HEK293 cells, selective activation of native STIM2 proteins or STIM2 overexpression results in store-operated activation of Imin channels, whereas STIM1 activation blocks this process. Changes in the ratio between active STIM2 and STIM1 proteins can switch the regulation of Imin channels between store-operated and store-independent modes. We have previously characterized electrophysiological properties of different Ca(2+) influx channels coexisting in HEK293 cells. The results of this study show that STIM1 and STIM2 differ in the ability to activate these store-operated channels; Imin channels are regulated by STIM2, TRPC3-containing INS channels are induced by STIM1, and TRPC1-composed Imax channels are activated by both STIM1 and STIM2. These new data about cross-talk between STIM1 and STIM2 and their different roles in store-operated channel activation are indicative of an additional level in the regulation of store-operated calcium entry pathways.

Store-operated calcium entry: unveiling the calcium handling signalplex.

Store-operated Ca(2+) entry (SOCE) is an important mechanism for Ca(2+) influx in non-excitable cells, also present in excitable cells. The activation of store-operated channels (SOCs) is finely regulated by the filling state of the intracellular agonist-sensitive Ca(2+) compartments, and both, the mechanism of sensing the Ca(2+) stores and the nature and functional properties of the SOCs, have been a matter of intense investigation and debate. The identification of STIM1 as the endoplasmic reticulum Ca(2+) sensorand both Orai1, as the pore-forming subunit of the channels mediating the Ca(2+)-selective store-operated current, and the members of the TRPC subfamily of proteins, as the channels mediating the cation-permeable SOCs, has shed new light on the underlying events. This review summarizes the initial hypothesis and the current advances on the mechanism of activation of SOCE.

STIM1 and STIM2 Proteins Regulation of Endogenous Store-Operated Calcium Channels in HEK293 Cells

The endoplasmic reticulum calcium sensors STIM1 and STIM2 are key modulators of store-operated calcium entry. They play a major role in physiological functions in the norm and pathology, however, available data on native STIM2-regulated plasma membrane channels are scarce: it is in only a few studies that STIM2-induced CRAC currents have been recorded. The STIM1 protein has been shown to regulate not only CRAC but also TRPC channels, but it has remained unclear whether STIM2 is capable of regulating store-operated non-CRAC channels. Here we present experimental evidence for the existence of endogenous non-CRAC STIM2-regulated channels. In single-channel patch-clamp experiments on HEK293 cells, store-operated Imin channels were induced by selective activation of native STIM2 proteins or STIM2 overexpression. It was found that STIM1 activation blocked store-operated mode of Imin activation. Changes in the ratio between active STIM2 and STIM1 proteins could switch Imin channels regulation between store-operated and store-independent modes.We have previously characterized electrophysiological properties of different Ca2+ influx channels coexisting in HEK293 cells. The results of this study show that STIM1 and STIM2 differ in the ability to activate these store-operated channels: Imin channels are regulated by STIM2, TRPC3-containing INS channels are induced by STIM1, and TRPC1-composed Imax channels are activated by both STIM1 and STIM2. These new data about cross-talk between STIM1 and STIM2 and their different roles in store-operated channel activation are indicative of an additional level in the regulation of store-operated calcium entry pathways.This study was supported by the Russian Scientific Foundation 14-14-00720 (to E.K. and A.S.); the program “Molecular and Cellular Biology” of the RAS (to G.N.M and L.G.); the Scientific School Support Program SS-1721.2014.4 (to G.N.M.); the Dynasty Foundation; and the Russian Foundation for Basic Research.

A relay mechanism between EB1 and APC facilitate STIM1 puncta assembly at endoplasmic reticulum–plasma membrane junctions

The assembly of STIM1 protein puncta near endoplasmic reticulum–plasma membrane (ER-PM) junctions is required for optimal activation of store-operated channels (SOC). The mechanisms controlling the translocation of STIM1 puncta to ER-PM junctions remain largely unknown.In the present study, we have explored the role of the microtubule binding protein adenomatous polyposis coli (APC), on STIM1 puncta and store-operated calcium entry (SOCE). APC-depleted cells showed reduced STIM1 puncta near ER-PM junctions, instead puncta is found at the ER surrounding the cell nucleus. Reduced STIM1 puncta near ER-PM junctions in APC-depleted cells correlates with a strong inhibition of SOCE and diminished Orai whole-cell currents. Immunoprecipitation and confocal microscopy co-localization studies indicate that, upon depletion of the ER, STIM1 dissociates from EB1 and associates to APC. Deletion analysis identified an APC-binding domain in the carboxyl terminus of STIM1 (STIM1 650–685).These results together position APC as an important element in facilitating the translocation of STIM1 puncta near ER-PM junctions, which in turn is required for efficient SOCE and Orai activation upon depletion of the ER.

Store-independent pathways for cytosolic STIM1 clustering in the regulation of store-operated Ca2+ influx

STIM1 is a Ca2+ sensing molecule. Once the Ca2+ stores are depleted, STIM1 moves towards the plasma membrane (PM) (translocation), forms puncta (clustering), and triggers store-operated Ca2+ entry (SOCE). Although this process has been regarded as a main mechanism for store-operated Ca2+ channel activation, the STIM1 clustering is still unclear. Here we discovered a new phenomenon of STIM1 clustering, which is not triggered by endoplasmic reticulum (ER) Ca2+ depletion.STIM1 subplasmalemmal translocation and clustering can be induced by ER Ca2+ store depletion with thapsigargin (TG), G-protein-coupled receptor activator trypsin and ryanodine receptor (RyR) agonists caffeine and 4-chloro-3-ethylphenol (4-CEP) in the HEK293 cells stably transfected with STIM1–EYFP. The STIM1 clustering induced by TG was more sustained than that induced by trypsin and RyR agonists. Interestingly, 4-CEP-induced STIM1 clustering also happened in the cytosol without ER Ca2+ store depletion. Application of some pharmacological regulators including flufenamic acid, 2-APB, and carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) at concentrations without affecting ER Ca2+ store also evoked cytosolic STIM1 clustering. However, the direct store-operated ORAI channel blockers (SKF-96365, Gd3+ and diethylstilbestrol) or the signaling pathway inhibitors (genistein, wortmannin, Y-27632, forskolin and GF109203X) did not change the STIM1 movement. Disruption of cytoskeleton by colchicine and cytochalasin D also showed no effect on STIM1 movement.We concluded that STIM1 clustering and translocation are two dynamic processes that can be pharmacologically dissociated. The ER Ca2+ store-independent mechanism for STIM1 clustering is a new alternative mechanism for regulating store-operated channel activity, which could act as a new pharmacological target.

Intracellular alkalinization induces cytosolic Ca2+ increases by inhibiting sarco/endoplasmic reticulum Ca2+-ATPase (SERCA).

Intracellular pH (pHi) and Ca2+ regulate essentially all aspects of cellular activities. Their inter-relationship has not been mechanistically explored. In this study, we used bases and acetic acid to manipulate the pHi. We found that transient pHi rise induced by both organic and inorganic bases, but not acidification induced by acid, produced elevation of cytosolic Ca2+. The sources of the Ca2+ increase are from the endoplasmic reticulum (ER) Ca2+ pools as well as from Ca2+ influx. The store-mobilization component of the Ca2+ increase induced by the pHi rise was not sensitive to antagonists for either IP3-receptors or ryanodine receptors, but was due to inhibition of the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA), leading to depletion of the ER Ca2+ store. We further showed that the physiological consequence of depletion of the ER Ca2+ store by pHi rise is the activation of store-operated channels (SOCs) of Orai1 and Stim1, leading to increased Ca2+ influx. Taken together, our results indicate that intracellular alkalinization inhibits SERCA activity, similar to thapsigargin, thereby resulting in Ca2+ leak from ER pools followed by Ca2+ influx via SOCs.

Hormone Signaling Linked to Silkmoth Sex Pheromone Biosynthesis Involves Ca2+/Calmodulin-dependent Protein Kinase II-mediated Phosphorylation of the Insect PAT Family Protein Bombyx mori Lipid Storage Droplet Protein-1 (BmLsd1)

Species-specific sex pheromones released by female moths to attract conspecific male moths are synthesized de novo in the pheromone gland (PG) via the fatty acid biosynthetic pathway. This pathway is regulated by a neurohormone termed pheromone biosynthesis activating neuropeptide (PBAN), a 33-amino acid peptide that originates in the subesophageal ganglion. In the silkmoth, Bombyx mori, cytoplasmic lipid droplets, which store the sex pheromone (bombykol) precursor fatty acid, accumulate in PG cells. PBAN stimulates lipolysis of the stored lipid droplet triacylglycerols (TAGs) and releases the precursor for final modification. PBAN exerts its physiological function via the PG cell-surface PBAN receptor, a G protein-coupled receptor that belongs to the neuromedin U receptor family. The PBAN receptor-mediated signal is transmitted via a canonical store-operated channel activation pathway utilizing Gq-mediated phospholipase C activation (Hull, J. J., Kajigaya, R., Imai, K., and Matsumoto, S. (2007) Biosci. Biotechnol. Biochem. 71, 1993-2001; Hull, J. J., Lee, J. M., Kajigaya, R., and Matsumoto, S. (2009) J. Biol. Chem. 284, 31200-31213; Hull, J. J., Lee, J. M., and Matsumoto, S. (2010) Insect Mol. Biol. 19, 553-566). Little, however, is known about the molecular components regulating TAG lipolysis in PG cells. In the current study we found that PBAN signaling involves phosphorylation of an insect PAT family protein named B. mori lipid storage droplet protein-1 (BmLsd1) and that BmLsd1 plays an essential role in the TAG lipolysis associated with bombykol production. Unlike mammalian PAT family perilipins, however, BmLsd1 activation is dependent on phosphorylation by B. mori Ca(2+)/calmodulin-dependent protein kinase II rather than protein kinase A.

Plasticity in Ca 2+ selectivity of Orai1/Orai3 heteromeric channel

A general cellular response following depletion of intracellular calcium stores involves activation of store-operated channels (SOCs). While Orai1 forms the native Ca(2+) release-activated Ca(2+) (CRAC) channel in mast and T cells, the molecular architecture of less Ca(2+) selective SOCs is insufficiently defined. Here we present evidence that diminished Ca(2+) selectivity and robust Cs(+) permeation together with a reduced fast inactivation are characteristics of heteromeric Orai1 and Orai3 channels in contrast to their homomeric forms. The first extracellular loop of these Orai isoforms differs by two aspartates replacing glutamates that affect the selectivity. Co-expression of an Orai3 mutant that mimicked the first loop of Orai1 with either Orai1 or Orai3 recovered or decreased Ca(2+) selectivity, respectively. Heteromeric Orai1/3 protein assembly provides a concept for less Ca(2+)-selective SOCs.

The role of homer family adaptor proteins in regulation of store-operated calcium influx in A431 cells

332 The major pathways for Ca 2+ influx in nonexcitable cells are store-operated channels (SOC). SOCs are activated by depletion of intracellular calcium stores. In the previous studies we used patch-clamp electrophysiology to describe the properties of store-operated channels I min in human carcinoma A431 cell lines. I min are gated by direct protein-protein interaction with inositol-1,4,5-trisphosphate receptor type 1 (IP 3 R1). We supposed that this interaction might be facilitated by scaffold proteins of Homer family. Homer family proteins specifically bind to unique PPXXF sequence in IP 3 R and in some other proteins. Several isoforms of Homer proteins exist. Homer 1c isoforms have coilcoiled domain, which mediate homooligomerization. That is why Homer 1c oligomers can form macromolecular complexes with partner proteins. Homer 1a isoform lacking coil-coiled domain can disrupt macromolecular complexes of Homer 1c and its partner proteins. The aim of this study was to determine the role of Homer protein in activation of store-operated channels. We applied the Gst-Homer recombinant protein (concentration 100 nM) to inside-out patches excised from A431 cells using 105 mM Ba 2+ in the pipette as a current carrier. Application of Gst-Homer 1c to the cytosolic surface of the patch did not activate channels. Addition of GST Homer 1a to the same patch resulted in an activation of I min channels.

Molecular Mechanisms Underlying PBAN Signaling in the Silkmoth Bombyx mori

Pheromone biosynthesis in the silkmoth Bombyx mori is under the control of the neurohormone pheromone biosynthesis activating neuropeptide (PBAN) and is triggered upon PBAN binding to its cognate PBAN receptor on the pheromone gland cells of female moths. Using fluorescent Ca(2+) imaging techniques with isolated pheromone glands, we have successfully demonstrated that PBAN specifically evokes an influx of extracellular Ca(2+). Furthermore, results from experiments designed to elucidate the molecular mechanisms underlying PBAN signaling indicate that B. mori utilizes the canonical store-operated channel-activation pathway and that the influx of extracellular Ca(2+) accelerates both lipolysis and fatty acyl reduction through a phosphorylation/dephosphorylation cascade for bombykol production.

Stopping STIM Stops Strokes

Proper responses of platelets in the blood to various physiological signals are essential: Unresponsiveness prevents clotting at sites of wounds, but overactivity can contribute to heart attacks or stroke. Platelets respond to a number of factors, but the various signals all feed into activation of phospholipase C and consequent release of calcium from intracellular stores. Depletion of calcium from the intracellular stores then leads to influx of extracellular calcium. The STIM1 protein (stromal interaction molecule 1) functions in detection of store emptying and activation of store-operated channels that allow calcium entry. Varga-Szabo et al . used knockout mice in which the Stim 1 gene was disrupted to study the role of STIM1 in arterial thrombosis and ischemic brain infarction. The Stim 1 –/– animals died a few hours after birth, but the authors were able to transplant bone marrow from the knockout animals to lethally irradiated host mice to study platelet function. In two in vivo models of arterial thrombosis induced by injury, formation of occlusive thrombi was inhibited in the Stim 1 –/– animals. Similarly, in a model of ischemic brain infarction induced by restriction of blood flow through the middle cerebral artery, infarct size was reduced in the Stim 1 –/– animals. Most animals with the Stim 1 –/– platelets stopped bleeding from a wound at the tail tip within about 10 minutes, as did wild-type mice. The response was more variable in the Stim 1 –/– animals, however, and about 35% of the animals showed longer bleeding. Because of this relatively mild effect on bleeding, the authors propose that inhibitors of Stim 1 might be attractive as antithrombolitics that could help treat stroke patients without the unacceptable side effect of increased intracerebral hemorrhage. As a caveat, the authors do note that results in the mouse models may not necessarily be directly comparable to the situation in humans. D. Varga-Szabo, A. Braun, C. Kleinschnitz, M. Bender, I. Pleines, M. Pham, T. Renné, G. Stoll, B. Nieswandt, The calcium sensor STIM1 is an essential mediator of arterial thrombosis and ischemic brain infarction. J. Exp. Med . 205 , 1583-1591 (2008). [Abstract] [Full Text]

Role of STIM and Orai proteins in the store-operated calcium signaling pathway

Ca 2+ signals are universal among cells in regulating a spectrum of cellular responses. Phospholipase C-coupled receptors activate two components of Ca 2+ signals—rapid Ca 2+ release from ER stores, followed by slower Ca 2+ entry from outside the cell. The coupling process between ER and PM to mediate this “store-operated” Ca 2+ entry process remained until recently a molecular mystery. The recent discovery of the necessity for STIM1 and Orai proteins in this process has provided crucial information on the coupling mechanism between stores and PM Ca 2+ entry. STIM1 is a single spanning membrane protein with an unpaired Ca 2+ binding EF-hand and appears to function as the sensor of ER luminal Ca 2+, and, through redistribution in the ER, transduces information directly to the PM. Orai1 is a tetra-spanning PM protein and functions as the highly Ca 2+-selective channel in the PM that is gated through interactions with the store-activated ER Ca 2+ sensor. Recent evidence shows the two proteins together are necessary and sufficient for the function of store-operated Ca 2+ entry. However, many questions arise about how and where the interactions of the STIM1 and Orai1 proteins occur within cells. Here we discuss recent information and ideas about the coupling between these proteins that leads to store-operated channel activation.

The Role of the Calcium Sensing Receptor in Regulating Intracellular Calcium Handling in Human Epidermal Keratinocytes

Calcium is critical for controlling the balance of proliferation and differentiation in epidermal keratinocytes. We previously reported that the calcium sensing receptor (CaR) is required for mediating Ca2+ signaling and extracellular Ca2+ (Ca2+(o))-induced differentiation. In this study, we investigated the mechanism by which CaR regulates intracellular Ca2+ (Ca2+(i)) and its role in differentiation. Membrane fractionation, fluorescence immunolocalization, and co-immunoprecipitation studies were performed to assess potential interactions between CaR and other regulators of Ca2+ stores and channels. We found that the glycosylated form of CaR forms a complex with phospholipase C gamma1, IP3 receptor (IP3R), and the Golgi Ca2+-ATPase, secretory pathway Ca2+-ATPase 1, in the trans-Golgi. Inactivation of the endogenous CaR gene by adenoviral expression of a CaR antisense cDNA inhibited Ca2+(i) response to Ca2+(o), decreased Ca2+(i) stores, decreased Ca2+(o)-induced differentiation, but augmented store-operated channel activity and Ca2+ uptake by intracellular organelles. Our results indicate that CaR regulates keratinocyte differentiation in part by modulating Ca2+(i) stores via interactions with Ca2+ pumps and channels that regulate those stores.

Recent breakthroughs in the molecular mechanism of capacitative calcium entry (with thoughts on how we got here)

Activation of phospholipase C by G-protein-coupled receptors results in release of intracellular Ca 2+ and activation of Ca 2+ channels in the plasma membrane. The intracellular release of Ca 2+ is signaled by the second messenger, inositol 1,4,5-trisphosphate. Ca 2+ entry involves signaling from depleted intracellular stores to plasma membrane Ca 2+ channels, a process referred to as capacitative calcium entry or store-operated calcium entry. The electrophysiological current associated with capacitative calcium entry is the calcium-release-activated calcium current, or I crac. In the 20 years since the inception of the concept of capacitative calcium entry, a variety of activation mechanisms have been proposed, and there has been considerable interest in the possibility of transient receptor potential channels functioning as store-operated channels. However, in the past 2 years, two major players in both the signaling and permeation mechanisms for store-operated channels have been discovered: Stim1 (and possibly Stim2) and the Orai proteins. Activation of store-operated channels involves an endoplasmic reticulum Ca 2+ sensor called Stim1. Stim1 acts by redistributing within a small component of the endoplasmic reticulum, approaching the plasma membrane, but does not appear to translocate into the plasma membrane. Stim1, either directly or indirectly, signals to plasma membrane Orai proteins which constitute pore-forming subunits of store-operated channels.

Calcium Entry Mechanisms in Salivary Glands

The organic and inorganic milieu of the oral cavity is regulated in large part through the secretions of salivary glands. These secretions are activated by a number of cellular signaling mechanisms, a major one being the cellular Ca2+ signaling system. Activation of phospholipase C by G-protein-coupled receptors results in release of intracellular Ca2+ and activation of Ca2+ channels in the plasma membrane. The intracellular release of Ca2+ is signaled by the second messenger, inositol 1,4,5-trisphosphate. Ca2+ entry involves signaling from depleted intracellular stores to plasma membrane Ca2+ channels, a process referred to as capacitative calcium entry or store-operated calcium entry. The electrophysiological current associated with capacitative calcium entry is the calcium-release-activated calcium current, or Icrac. In the twenty years since the inception of the concept of capacitative calcium entry, a variety of activation mechanisms have been proposed, and there has been considerable interest in the possibility of transient receptor potential channels functioning as store-operated channels. However, in the past two years, two major players in both the signaling and permeation mechanisms for store-operated channels have been discovered : Stim1 (and possibly Stim2) and the Orai proteins. Activation of store-operated channels involves an endoplasmic reticulum Ca2+ sensor called Stim1. Stim1 acts by redistributing within a small component of the endoplasmic reticulum, approaching the plasma membrane, but does not appear to translocate into the plasma membrane. Stim1, either directly or indirectly, signals to plasma membrane Orai proteins which constitute pore-forming subunits of store-operated channels.

Priming of long-term potentiation mediated by ryanodine receptor activation in rat hippocampal slices

Administration of the Group 1 metabotropic glutamate receptor (mGluR) agonist ( R,S)-3,5-dihydroxyphenylglycine (DHPG) facilitates (“primes”) subsequent long-term potentiation (LTP) through a phospholipase C signaling cascade that may involve release of Ca 2+ from the endoplasmic reticulum (ER). We investigated the intracellular calcium pathways involved in this priming effect, recording field potentials from area CA1 of rat hippocampal slices before and after high-frequency stimulation. The priming of LTP by DHPG was prevented by co-administration of cyclopiazonic acid, which depletes ER Ca 2+ stores. The priming effect was also blocked by the ryanodine receptor (RYR) antagonist ryanodine (RYA, 100 μM). In contrast, a low dose of RYA (10 μM) which opens the RYR channel, by itself primed LTP. In addition to RYR activation, entry of extracellular calcium through store-operated channels appears necessary for priming, since diverse treatments known to impede store-operated channel activity completely blocked both RYA and DHPG priming effects. Thus, RYR activation plays a critical role in the priming of LTP by Group 1 mGluRs, and this effect is coupled to the entry of extracellular calcium, probably through store-operated calcium channels.

Orai1 and STIM Reconstitute Store-operated Calcium Channel Function

The two membrane proteins, STIM1 and Orai1, have each been shown to be essential for the activation of store-operated channels (SOC). Yet, how these proteins functionally interact is not known. Here, we reveal that STIM1 and Orai1 expressed together reconstitute functional SOCs. Expressed alone, Orai1 strongly reduces store-operated Ca(2+) entry (SOCE) in human embryonic kidney 293 cells and the Ca(2+) release-activated Ca(2+) current (I(CRAC)) in rat basophilic leukemia cells. However, expressed along with the store-sensing STIM1 protein, Orai1 causes a massive increase in SOCE, enhancing the rate of Ca(2+)entry by up to 103-fold. This entry is entirely store-dependent since the same coexpression causes no measurable store-independent Ca(2+) entry. The entry is completely blocked by the SOC blocker, 2-aminoethoxydiphenylborate. Orai1 and STIM1 coexpression also caused a large gain in CRAC channel function in rat basophilic leukemia cells. The close STIM1 homologue, STIM2, inhibited SOCE when expressed alone but coexpressed with Orai1 caused substantial constitutive (store-independent) Ca(2+) entry. STIM proteins are known to mediate Ca(2+) store-sensing and endoplasmic reticulum-plasma membrane coupling with no intrinsic channel properties. Our results revealing a powerful gain in SOC function dependent on the presence of both Orai1 and STIM1 strongly suggest that Orai1 contributes the PM channel component responsible for Ca(2+) entry. The suppression of SOC function by Orai1 overexpression likely reflects a required stoichiometry between STIM1 and Orai1.

Passive calcium leak via translocon is a first step for iPLA2‐pathway regulated store operated channels activation

Calcium concentration within the endoplasmic reticulum (ER) plays an essential role in cell physiopathology. One of the most enigmatic mechanisms responsible for Ca2+ concentration in the ER is passive calcium leak. Previous studies have shown that the translocon complex is permeable to calcium. However, the involvement of the translocon in the passive calcium leak has not been directly demonstrated. Furthermore, the question whether the passive store depletion via the translocon could activate SOC (store operated channels) replenishing the ER, remains still unresolved. In this study, for the first time, we show that thapsigargin and calcium chelators deplete ER via translocon. Indeed, using confocal imaging, we demonstrate that when the number of opened translocons was lowered neither thapsigargin nor calcium chelators could induce ER store depletion. We also demonstrate that calcium leakage occurring via the translocon activates store-operated current, which is, by its kinetic and pharmacology, similar to that evoked by thapsigargin and EGTA (but not IP3), thus highlighting our hypothesis that calcium leakage via the translocon is a first step for activation of the specific iPLA2-regulated SOC. As the translocon is present in yeast and mammalian cells, our findings suggest that translocon-related calcium signaling is a common phenomenon.

GABAB heterodimeric receptors promote Ca2+ influx via store-operated channels in rat cortical neurons and transfected chinese hamster ovary cells

The GABAB receptors are generally considered to be classical Gi-coupled receptors that lack the ability to mobilize intracellular Ca2+ without the aid of promiscuous G proteins. Here, we report the ability of GABAB receptors to promote calcium influx into primary cultures of rat cortical neurons and transfected Chinese hamster ovary cells. Chinese hamster ovary cells were transfected with GABAB1(a) or GABAB1(b) subunits along with GABAB2 subunits. In experiments using the fluorometric imaging plate reader platform, GABA and selective agonists promoted increases in intracellular Ca2+ levels in transfected Chinese hamster ovary cells and cortical neurons with the expected order of potency. These effects were fully antagonized by selective GABAB receptor antagonists. To investigate the intracellular pathways responsible for mediating these effects we employed several pharmacological inhibitors. Pertussis toxin abolished GABAB mediated Ca2+ increases, as did the phospholipase Cβ inhibitor U73122. Inhibitor 2-aminethoxydiphenyl borane acts as an antagonist at inositol 1,4,5-trisphosphate receptors and at store-operated channels. In all cell types, 2-aminethoxydiphenyl borane prevented Ca2+ mobilization. The selective store-operated channel inhibitor 1-[2-(4-methoxyphenyl)-2-[3-(4-methoxyphenyl)propoxy]ethyl-1H-imidazole hydrochloride prevented increases in intracellular Ca2+ levels as did performing the assays in Ca2+ free buffers. In conclusion, GABAB receptors expressed in Chinese hamster ovary cells and endogenously expressed in rat cortical neurons promote Ca2+ entry into the cell via the activation of store-operated channels, using a mechanism that is dependent on Gi/o heterotrimeric proteins and phospholipase Cβ. These findings suggest that the neuronal effects mediated by GABAB receptors may, in part, rely on the receptor’s ability to promote Ca2+ influx.