Scrutinizing science to save lives: uncovering flaws in the data linking L-type calcium channels blockers to CRAC channels and heart failure.

Hypertension is estimated to affect almost 1 billion people globally and significantly increases risk of myocardial infarction, heart failure, stroke, retinopathy and kidney disease. One major front line therapy that has been used for over 50 years involves L-type Ca 2+ channel blockers (LCCBs). One class of LCCBs is the dihydropyridine family, with amlodipine being widely prescribed regardless of gender, race, ethnicity or age. In 2020, Johnson et al. 7 reported that all LCCBs significantly increased the risk of heart failure, and attributed this effect to non-canonical activation of store-operated Ca 2+ entry. A major approach on which they based many of their arguments was to measure cytosolic Ca 2+ using the fluorescent Ca 2+ indicator dye fura-2. We recently demonstrated that amlodipine is highly fluorescent within cells and overwhelms the fura-2 signal, precluding the use of the indicator dye with amlodipine 24 . Our meta-analyses and prospective real world study showed that dihydropyridines were not associated with an increase in heart failure, likely explained by the lack of consideration by Johnson et al. 7 of well-known confounding factors such as age, race, obesity, prior anti-hypertensive treatment or diabetes 24 . Trebak and colleagues have responded to our paper with a forthright and unwavering defence of their work 27 . In this paper, we carry out a forensic dissection of Johnson et al., 7 and conduct new experiments that address directly points raised by Trebak et al. 27 . We show that there are major flaws in the design and interpretation of their key experiments, that fura-2 cannot be used with amlodipine, that there are fundamental mathematical misunderstandings and mistakes throughout their study leading to critical calculations on heart failure that are demonstrably wrong, and several of their own results are inconsistent with their interpretation. We therefore believe the study by Johnson et al. 7 is flawed at many levels and we stand by our conclusions.

A Reappraisal of the Effects of L-type Ca2+ Channel Blockers on Store-Operated Ca2+ Entry and Heart Failure.

Dihydropyridines such as amlodipine are widely used as antihypertensive agents, being prescribed to ∼70 million Americans and >0.4 billion adults worldwide. Dihydropyridines block voltage-gated Ca2+ channels in resistance vessels, leading to vasodilation and a reduction in blood pressure. Various meta-analyses show that dihydropyridines are relatively safe and effective in reducing hypertension. The use of dihydropyridines has recently been called into question as these drugs appear to activate store-operated Ca2+ entry in fura-2-loaded nonexcitable cells, trigger vascular remodeling, and increase heart failure, leading to the questioning of their clinical use. Given that hypertension is the dominant "silent killer" across the globe affecting ∼1.13 billion people, removal of Ca2+ channel blockers as antihypertensive agents has major health implications. Here, we show that amlodipine has marked intrinsic fluorescence, which further increases considerably inside cells over an identical excitation spectrum as fura-2, confounding the ability to measure cytosolic Ca2+. Using longer wavelength Ca2+ indicators, we find that concentrations of Ca2+ channel blockers that match therapeutic levels in serum of patients do not activate store-operated Ca2+ entry. Antihypertensive Ca2+ channel blockers at pharmacological concentrations either have no effect on store-operated channels, activate them indirectly through store depletion or inhibit the channels. Importantly, a meta-analysis of published clinical trials and a prospective real-world analysis of patients prescribed single antihypertensive agents for 6 mo and followed up 1 yr later both show that dihydropyridines are not associated with increased heart failure or other cardiovascular disorders. Removal of dihydropyridines for treatment of hypertension cannot therefore be recommended.

The N terminus of Orai1 couples to the AKAP79 signaling complex to drive NFAT1 activation by local Ca2+ entry.

To avoid conflicting and deleterious outcomes, eukaryotic cells often confine second messengers to spatially restricted subcompartments. The smallest signaling unit is the Ca2+ nanodomain, which forms when Ca2+ channels open. Ca2+ nanodomains arising from store-operated Orai1 Ca2+ channels stimulate the protein phosphatase calcineurin to activate the transcription factor nuclear factor of activated T cells (NFAT). Here, we show that NFAT1 tethered directly to the scaffolding protein AKAP79 (A-kinase anchoring protein 79) is activated by local Ca2+ entry, providing a mechanism to selectively recruit a transcription factor. We identify the region on the N terminus of Orai1 that interacts with AKAP79 and demonstrate that this site is essential for physiological excitation-transcription coupling. NMR structural analysis of the AKAP binding domain reveals a compact shape with several proline-driven turns. Orai2 and Orai3, isoforms of Orai1, lack this region and therefore are less able to engage AKAP79 and activate NFAT. A shorter, naturally occurring Orai1 protein that arises from alternative translation initiation also lacks the AKAP79-interaction site and fails to activate NFAT1. Interfering with Orai1-AKAP79 interaction suppresses cytokine production, leaving other Ca2+ channel functions intact. Our results reveal the mechanistic basis for how a subtype of a widely expressed Ca2+ channel is able to activate a vital transcription pathway and identify an approach for generation of immunosuppressant drugs.

Store-operated Ca2+ entry and Ca2+ responses to hypothalamic releasing hormones in anterior pituitary cells from Orai1-/- and heptaTRPC knockout mice.

Store operated Ca2+ entry (SOCE) is the most important Ca2+ entry pathway in non-excitable cells. However, SOCE can also play a pivotal role in excitable cells such as anterior pituitary (AP) cells. The AP gland contains five different cell types that release six major AP hormones controlling most of the entire endocrine system. AP hormone release is modulated by Ca2+ signals induced by different hypothalamic releasing hormones (HRHs) acting on specific receptors in AP cells. TRH and LHRH both induce Ca2+ release and Ca2+ entry in responsive cells while GHRH and CRH only induce Ca2+ entry. SOCE has been shown to contribute to Ca2+ responses induced by TRH and LHRH but no molecular evidence has been provided. Accordingly, we used AP cells isolated from mice devoid of Orai1 channels (noted as Orai1-/- or Orai1 KO mice) and mice lacking expression of all seven canonical TRP channels (TRPC) from TRPC1 to TRPC7 (noted as heptaTRPC KO mice) to investigate contribution of these putative channel proteins to SOCE and intracellular Ca2+ responses induced by HRHs. We found that thapsigargin-evoked SOCE is lost in AP cells from Orai1-/- mice but unaffected in cells from heptaTRPC KO mice. Conversely, while spontaneous intracellular Ca2+-oscillations related to electrical activity were not affected in the Orai1-/- mice, these responses were significantly reduced in heptaTRPC KO mice. We also found that Ca2+ entry induced by TRH and LHRH is decreased in AP cells isolated from Orai1-/-. In addition, Ca2+ responses to several HRHs, particularly TRH and GHRH, are decreased in the heptaTRPC KO mice. These results indicate that expression of Orai1, and not TRPC channel proteins, is necessary for thapsigargin-evoked SOCE and is required to support Ca2+ entry induced by TRH and LHRH in mouse AP cells. In contrast, TRPC channel proteins appear to contribute to spontaneous Ca2+-oscillations and Ca2+ responses induced by TRH and GHRH. We conclude that expression of Orai1 and TRPC channels proteins may play differential and significant roles in AP physiology and endocrine control.

Cytokine signaling through Drosophila Mthl10 ties lifespan to environmental stress.

A systems-level understanding of cytokine-mediated, intertissue signaling is one of the keys to developing fundamental insight into the links between aging and inflammation. Here, we employed Drosophila, a routine model for analysis of cytokine signaling pathways in higher animals, to identify a receptor for the growth-blocking peptide (GBP) cytokine. Having previously established that the phospholipase C/Ca2+ signaling pathway mediates innate immune responses to GBP, we conducted a dsRNA library screen for genes that modulate Ca2+ mobilization in Drosophila S3 cells. A hitherto orphan G protein coupled receptor, Methuselah-like receptor-10 (Mthl10), was a significant hit. Secondary screening confirmed specific binding of fluorophore-tagged GBP to both S3 cells and recombinant Mthl10-ectodomain. We discovered that the metabolic, immunological, and stress-protecting roles of GBP all interconnect through Mthl10. This we established by Mthl10 knockdown in three fly model systems: in hemocyte-like Drosophila S2 cells, Mthl10 knockdown decreases GBP-mediated innate immune responses; in larvae, Mthl10 knockdown decreases expression of antimicrobial peptides in response to low temperature; in adult flies, Mthl10 knockdown increases mortality rate following infection with Micrococcus luteus and reduces GBP-mediated secretion of insulin-like peptides. We further report that organismal fitness pays a price for the utilization of Mthl10 to integrate all of these various homeostatic attributes of GBP: We found that elevated GBP expression reduces lifespan. Conversely, Mthl10 knockdown extended lifespan. We describe how our data offer opportunities for further molecular interrogation of yin and yang between homeostasis and longevity.

The functions of store-operated calcium channels.

Store-operated calcium channels provide calcium signals to the cytoplasm of a wide variety of cell types. The basic components of this signaling mechanism include a mechanism for discharging Ca2+ stores (commonly but not exclusively phospholipase C and inositol 1,4,5-trisphosphate), a sensor in the endoplasmic reticulum that also serves as an activator of the plasma membrane channel (STIM1 and STIM2), and the store-operated channel (Orai1, 2 or 3). The advent of mice genetically altered to reduce store-operated calcium entry globally or in specific cell types has provided important tools to understand the functions of these widely encountered channels in specific and clinically important physiological systems. This review briefly discusses the history and cellular properties of store-operated calcium channels, and summarizes selected studies of their physiological functions in specific physiological or pathological contexts. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.

Male infertility in mice lacking the store-operated Ca(2+) channel Orai1.

Store-operated calcium entry (SOCE) is an important Ca(2+) influx pathway in somatic cells. In addition to maintaining endoplasmic reticulum (ER) Ca(2+) stores, Ca(2+) entry through store-operated channels regulates essential signaling pathways in numerous cell types. Patients with mutations in the store-operated channel subunit ORAI1 exhibit defects in store-operated Ca(2+) influx, along with severe immunodeficiency, congenital myopathy and ectodermal dysplasia. However, little is known about the functional role of ORAI1 in germ cells and reproductive function in mice, or in men, since men with loss-of-function or null mutations in ORAI1 rarely survive to reproductive age. In this study, we investigated the role of ORAI1 in male reproductive function. We reveal that Orai1(-/-) male mice are sterile and have severe defects in spermatogenesis, with prominent deficiencies in mid- to late-stage elongating spermatid development. These studies establish an essential in vivo role for store-operated ORAI1 channels in male reproductive function and identify these channels as potential non-steroidal regulators of male fertility.

Essential role of Orai1 store-operated calcium channels in lactation.

The nourishment of neonates by nursing is the defining characteristic of mammals. However, despite considerable research into the neural control of lactation, an understanding of the signaling mechanisms underlying the production and expulsion of milk by mammary epithelial cells during lactation remains largely unknown. Here we demonstrate that a store-operated Ca(2+) channel subunit, Orai1, is required for both optimal Ca(2+) transport into milk and for milk ejection. Using a novel, 3D imaging strategy, we visualized live oxytocin-induced alveolar unit contractions in the mammary gland, and we demonstrated that in this model milk is ejected by way of pulsatile contractions of these alveolar units. In mammary glands of Orai1 knockout mice, these contractions are infrequent and poorly coordinated. We reveal that oxytocin also induces a large transient release of stored Ca(2+) in mammary myoepithelial cells followed by slow, irregular Ca(2+) oscillations. These oscillations, and not the initial Ca(2+) transient, are mediated exclusively by Orai1 and are absolutely required for milk ejection and pup survival, an observation that redefines the signaling processes responsible for milk ejection. These findings clearly demonstrate that Ca(2+) is not just a substrate for nutritional enrichment in mammals but is also a master regulator of the spatiotemporal signaling events underpinning mammary alveolar unit contraction. Orai1-dependent Ca(2+) oscillations may represent a conserved language in myoepithelial cells of other secretory epithelia, such as sweat glands, potentially shedding light on other Orai1 channelopathies, including anhidrosis (an inability to sweat).

Switching between humoral and cellular immune responses in Drosophila is guided by the cytokine GBP.

Insects combat infection through carefully measured cellular (for example, phagocytosis) and humoral (for example, secretion of antimicrobial peptides (AMPs)) innate immune responses. Little is known concerning how these different defense mechanisms are coordinated. Here, we use insect plasmatocytes and hemocyte-like Drosophila S2 cells to characterize mechanisms of immunity that operate in the haemocoel. We demonstrate that a Drosophila cytokine, growth-blocking peptides (GBP), acts through the phospholipase C (PLC)/Ca(2+) signalling cascade to mediate the secretion of Pvf, a ligand for platelet-derived growth factor- and vascular endothelial growth factor-receptor (Pvr) homologue. Activated Pvr recruits extracellular signal-regulated protein kinase to inhibit humoral immune responses, while stimulating cell 'spreading', an initiating event in cellular immunity. The double-stranded RNA (dsRNA)-targeted knockdown of either Pvf2 or Pvr inhibits GBP-mediated cell spreading and activates AMP expression. Conversely, Pvf2 overexpression enhances cell spreading but inhibits AMP expression. Thus, we describe mechanisms to initiate immune programs that are either humoral or cellular in nature, but not both; such immunophysiological polarization may minimize homeostatic imbalance during infection.

Calcium signaling in lacrimal glands.

Lacrimal glands provide the important function of lubricating and protecting the ocular surface. Failure of proper lacrimal gland function results in a number of debilitating dry eye diseases. Lacrimal glands secrete lipids, mucins, proteins, salts and water and these secretions are at least partially regulated by neurotransmitter-mediated cell signaling. The predominant signaling mechanism for lacrimal secretion involves activation of phospholipase C, generation of the Ca(2+)-mobilizing messenger, IP3, and release of Ca(2+) stored in the endoplasmic reticulum. The loss of Ca(2+) from the endoplasmic reticulum then triggers a process known as store-operated Ca(2+) entry, involving a Ca(2+) sensor in the endoplasmic reticulum, STIM1, which activates plasma membrane store-operated channels comprised of Orai subunits. Recent studies with deletions of the channel subunit, Orai1, confirm the important role of SOCE in both fluid and protein secretion in lacrimal glands, both in vivo and in vitro.

Role of Orai1 and store-operated calcium entry in mouse lacrimal gland signalling and function.

Lacrimal glands function to produce an aqueous layer, or tear film, that helps to nourish and protect the ocular surface. Lacrimal glands secrete proteins, electrolytes and water, and loss of gland function can result in tear film disorders such as dry eye syndrome, a widely encountered and debilitating disease in ageing populations. To combat these disorders, understanding the underlying molecular signalling processes that control lacrimal gland function will give insight into corrective therapeutic approaches. Previously, in single lacrimal cells isolated from lacrimal glands, we demonstrated that muscarinic receptor activation stimulates a phospholipase C-coupled signalling cascade involving the inositol trisphosphate-dependent mobilization of intracellular calcium and the subsequent activation of store-operated calcium entry (SOCE). Since intracellular calcium stores are finite and readily exhausted, the SOCE pathway is a critical process for sustaining and maintaining receptor-activated signalling. Recent studies have identified the Orai family proteins as critical components of the SOCE channel activity in a wide variety of cell types. In this study we characterize the role of Orai1 in the function of lacrimal glands using a mouse model in which the gene for the calcium entry channel protein, Orai1, has been deleted. Our data demonstrate that lacrimal acinar cells lacking Orai1 do not exhibit SOCE following activation of the muscarinic receptor. In comparison with wild-type and heterozygous littermates, Orai1 knockout mice showed a significant reduction in the stimulated tear production following injection of pilocarpine, a muscarinic receptor agonist. In addition, calcium-dependent, but not calcium-independent exocytotic secretion of peroxidase was eliminated in glands from knockout mice. These studies indicate a critical role for Orai1-mediated SOCE in lacrimal gland signalling and function.

Essential role of stress hormone signaling in cardiomyocytes for the prevention of heart disease.

Heart failure is a leading cause of death in humans, and stress is increasingly associated with adverse cardiac outcomes. Glucocorticoids are primary stress hormones, but their direct role in cardiovascular health and disease is poorly understood. To determine the in vivo function of glucocorticoid signaling in the heart, we generated mice with cardiomyocyte-specific deletion of the glucocorticoid receptor (GR). These mice are born at the expected Mendelian ratio, but die prematurely from spontaneous cardiovascular disease. By 3 mo of age, mice deficient in cardiomyocyte GR display a marked reduction in left ventricular systolic function, as evidenced by decreases in ejection fraction and fractional shortening. Heart weight and left ventricular mass are elevated, and histology revealed cardiac hypertrophy without fibrosis. Removal of endogenous glucocorticoids and mineralocorticoids neither augmented nor lessened the hypertrophic response. Global gene expression analysis of knockout hearts before pathology onset revealed aberrant regulation of a large cohort of genes associated with cardiovascular disease as well as unique disease genes associated with inflammatory processes. Genes important for maintaining cardiac contractility, repressing cardiac hypertrophy, promoting cardiomyocyte survival, and inhibiting inflammation had decreased expression in the GR-deficient hearts. These findings demonstrate that a deficiency in cardiomyocyte glucocorticoid signaling leads to spontaneous cardiac hypertrophy, heart failure, and death, revealing an obligate role for GR in maintaining normal cardiovascular function. Moreover, our findings suggest that selective activation of cardiomyocyte GR may represent an approach for the prevention of heart disease.

Deletion of Orai1 alters expression of multiple genes during osteoclast and osteoblast maturation.

Store-operated Ca(2+) entry (SOCE) is a major Ca(2+) influx pathway in most non-excitable cell types and Orai1 was recently identified as an essential pore-subunit of SOCE channels. Here we investigate the physiological role of Orai1 in bone homeostasis using Orai1-deficient mice (Orai1(-/-)). Orai1(-/-) mice developed osteopenia with decreased bone mineral density and trabecular bone volume. To identify the nature and origin of the bone defect, bone-resorbing osteoclasts and bone-forming osteoblasts from Orai1(-/-) mice were examined. Orai1-mediated SOCE was completely abolished in Orai1(-/-) osteoclast precursor cells and osteoclastogenesis in vitro from Orai1(-/-) mice was impaired due to a defect in cell fusion of pre-osteoclasts. Also, resorption activity in vitro was comparable but the size of pits formed by Orai1(-/-) osteoclasts was smaller. We next assessed the role of Orai1 in osteoblast differentiation and function by using a pre-osteoblast cell line, as well as primary osteoblasts from wild-type and Orai1(-/-) mice. SOCE in MC3T3-E1 pre-osteoblastic cells was inactivated by lentiviral overexpression of a pore-dead Orai1 mutant. Lack of SOCE in MC3T3-E1 had no effect on alkaline phosphatase staining and expression but substantially inhibited mineralized nodule formation. Consistent with this finding, Orai1-mediated SOCE was markedly reduced in Orai1(-/-) osteoblast precursor cells and osteoblastogenesis in vitro from Orai1(-/-) stromal cells showed impaired mineral deposition but no change in differentiation. This indicates that Orai1 is involved in the function but not in the differentiation of osteoblasts. Together, these results suggest that Orai1 plays a critical role in bone homeostasis by regulating both osteoblasts and osteoclasts.

Activation of PLC by an endogenous cytokine (GBP) in Drosophila S3 cells and its application as a model for studying inositol phosphate signalling through ITPK1.

Using immortalized [3H]inositol-labelled S3 cells, we demonstrated in the present study that various elements of the inositol phosphate signalling cascade are recruited by a Drosophila homologue from a cytokine family of so-called GBPs (growth-blocking peptides). HPLC analysis revealed that dGBP (Drosophila GBP) elevated Ins(1,4,5)P3 levels 9-fold. By using fluorescent Ca2+ probes, we determined that dGBP initially mobilized Ca2+ from intracellular pools; the ensuing depletion of intracellular Ca2+ stores by dGBP subsequently activated a Ca2+ entry pathway. The addition of dsRNA (double-stranded RNA) to knock down expression of the Drosophila Ins(1,4,5)P3 receptor almost completely eliminated mobilization of intracellular Ca2+ stores by dGBP. Taken together, the results of the present study describe a classical activation of PLC (phospholipase C) by dGBP. The peptide also promoted increases in the levels of other inositol phosphates with signalling credentials: Ins(1,3,4,5)P4, Ins(1,4,5,6)P4 and Ins(1,3,4,5,6)P5. These results greatly expand the regulatory repertoire of the dGBP family, and also characterize S3 cells as a model for studying the regulation of inositol phosphate metabolism and signalling by endogenous cell-surface receptors. We therefore created a cell-line (S3ITPK1) in which heterologous expression of human ITPK (inositol tetrakisphosphate kinase) was controlled by an inducible metallothionein promoter. We found that dGBP-stimulated S3ITPK1 cells did not synthesize Ins(3,4,5,6)P4, contradicting a hypothesis that the PLC-coupled phosphotransferase activity of ITPK1 [Ins(1,3,4,5,6)P5+Ins(1,3,4)P3→Ins(3,4,5,6)P4+Ins(1,3,4,6)P4] is driven solely by the laws of mass action [Chamberlain, Qian, Stiles, Cho, Jones, Lesley, Grabau, Shears and Spraggon (2007) J. Biol. Chem. 282, 28117-28125]. This conclusion represents a fundamental breach in our understanding of ITPK1 signalling.

Cadmium induces transcription independently of intracellular calcium mobilization.

BACKGROUND: Exposure to cadmium is associated with human pathologies and altered gene expression. The molecular mechanisms by which cadmium affects transcription remain unclear. It has been proposed that cadmium activates transcription by altering intracellular calcium concentration ([Ca(2+)](i)) and disrupting calcium-mediated intracellular signaling processes. This hypothesis is based on several studies that may be technically problematic; including the use of BAPTA chelators, BAPTA-based fluorescent sensors, and cytotoxic concentrations of metal. METHODOLOGY/PRINCIPAL FINDING: In the present report, the effects of cadmium on [Ca(2+)](i) under non-cytotoxic and cytotoxic conditions was monitored using the protein-based calcium sensor yellow cameleon (YC3.60), which was stably expressed in HEK293 cells. In HEK293 constitutively expressing YC3.60, this calcium sensor was found to be insensitive to cadmium. Exposing HEK293::YC3.60 cells to non-cytotoxic cadmium concentrations was sufficient to induce transcription of cadmium-responsive genes but did not affect [Ca(2+)](i) mobilization or increase steady-state mRNA levels of calcium-responsive genes. In contrast, exposure to cytotoxic concentrations of cadmium significantly reduced intracellular calcium stores and altered calcium-responsive gene expression. CONCLUSIONS/SIGNIFICANCE: These data indicate that at low levels, cadmium induces transcription independently of intracellular calcium mobilization. The results also support a model whereby cytotoxic levels of cadmium activate calcium-responsive transcription as a general response to metal-induced intracellular damage and not via a specific mechanism. Thus, the modulation of intracellular calcium may not be a primary mechanism by which cadmium regulates transcription.

Amantadine protects dopamine neurons by a dual action: reducing activation of microglia and inducing expression of GDNF in astroglia [corrected].

Amantadine is commonly given to alleviate L-DOPA-induced dyskinesia of Parkinson's disease (PD) patients. Animal and human evidence showed that amantadine may also exert neuroprotection in several neurological disorders. Additionally, it is generally believed that this neuroprotection results from the ability of amantadine to inhibit glutamatergic NMDA receptor. However, several lines of evidence questioned the neuroprotective capacity of NMDA receptor antagonists in animal models of PD. Thus the cellular and molecular mechanism of neuroprotection of amantadine remains unclear. Using primary cultures with different composition of neurons, microglia, and astroglia we investigated the direct role of these glial cell types in the neuroprotective effect of amantadine. First, amantadine protected rat midbrain cultures from either MPP(+) or lipopolysaccharide (LPS), two toxins commonly used as PD models. Second, our studies revealed that amantadine reduced both LPS- and MPP(+)-induced toxicity of dopamine neurons through 1) the inhibition of the release of microglial pro-inflammatory factors, 2) an increase in expression of neurotrophic factors such as GDNF from astroglia. Lastly, differently from the general view on amantadine's action, we provided evidence suggesting that NMDA receptor inhibition was not crucial for the neuroprotective effect of amantadine. In conclusion, we report that amantadine protected dopamine neurons in two PD models through a novel dual mechanism, namely reducing the release of pro-inflammatory factors from activated microglia and increasing the expression of GNDF in astroglia.

Calcium oscillations.

Calcium signaling results from a complex interplay between activation and inactivation of intracellular and extracellular calcium permeable channels. This complexity is obvious from the pattern of calcium signals observed with modest, physiological concentrations of calcium-mobilizing agonists, which typically present as sequential regenerative discharges of stored calcium, a process referred to as calcium oscillations. In this review, we discuss recent advances in understanding the underlying mechanism of calcium oscillations through the power of mathematical modeling. We also summarize recent findings on the role of calcium entry through store-operated channels in sustaining calcium oscillations and in the mechanism by which calcium oscillations couple to downstream effectors.

STIM1 is a calcium sensor specialized for digital signaling.

When cells are activated by calcium-mobilizing agonists at low, physiological concentrations, the resulting calcium signals generally take the form of repetitive regenerative discharges of stored calcium, termed calcium oscillations [1]. These intracellular calcium oscillations have long fascinated biologists as a mode of digitized intracellular signaling. Recent work has highlighted the role of calcium influx as an essential component of calcium oscillations [2]. This influx occurs through a process known as store-operated calcium entry, which is initiated by calcium sensor proteins, STIM1 and STIM2, in the endoplasmic reticulum [3]. STIM2 is activated by changes in endoplasmic reticulum calcium near the resting level, whereas a threshold of calcium depletion is required for STIM1 activation [4]. Here we show that, surprisingly, it is STIM1 and not STIM2 that is exclusively involved in calcium entry during calcium oscillations. The implication is that each oscillation produces a transient drop in endoplasmic reticulum calcium and that this drop is sufficient to transiently activate STIM1. This transient activation of STIM1 can be observed in some cells by total internal reflection fluorescence microscopy. This arrangement nicely provides a clearly defined and unambiguous signaling system, translating a digital calcium release signal into calcium influx that can signal to downstream effectors.

TRPC channels function independently of STIM1 and Orai1.

Recent studies have defined roles for STIM1 and Orai1 as calcium sensor and calcium channel, respectively, for Ca(2+)-release activated Ca(2+) (CRAC) channels, channels underlying store-operated Ca(2+) entry (SOCE). In addition, these proteins have been suggested to function in signalling and constructing other channels with biophysical properties distinct from the CRAC channels. Using the human kidney cell line, HEK293, we examined the hypothesis that STIM1 can interact with and regulate members of a family of non-selective cation channels (TRPC) which have been suggested to also function in SOCE pathways under certain conditions. Our data reveal no role for either STIM1 or Orai1 in signalling of TRPC channels. Specifically, Ca(2+) entry seen after carbachol treatment in cells transiently expressing TRPC1, TRPC3, TRPC5 or TRPC6 was not enhanced by the co-expression of STIM1. Further, knockdown of STIM1 in cells expressing TRPC5 did not reduce TRPC5 activity, in contrast to one published report. We previously reported in stable TRPC7 cells a Ca(2+) entry which was dependent on TRPC7 and appeared store-operated. However, we show here that this TRPC7-mediated entry was also not dependent on either STIM1 or Orai1, as determined by RNA interference (RNAi) and expression of a constitutively active mutant of STIM1. Further, we determined that this entry was not actually store-operated, but instead TRPC7 activity which appears to be regulated by SERCA. Importantly, endogenous TRPC activity was also not regulated by STIM1. In vascular smooth muscle cells, arginine-vasopressin (AVP) activated non-selective cation currents associated with TRPC6 activity were not affected by RNAi knockdown of STIM1, while SOCE was largely inhibited. Finally, disruption of lipid rafts significantly attenuated TRPC3 activity, while having no effect on STIM1 localization or the development of I(CRAC). Also, STIM1 punctae were found to localize in regions distinct from lipid rafts. This suggests that TRPC signalling and STIM1/Orai1 signalling occur in distinct plasma membrane domains. Thus, TRPC channels appear to be activated by mechanisms dependent on phospholipase C which do not involve the Ca(2+) sensor, STIM1.