PtdSer as a signaling lipid determined by privileged localization of ORP5 and ORP8 at ER/PM junctional foci to determine PM and ER PtdSer/PI(4)P ratio and cell function.

The membrane contact site ER/PM junctions are hubs for signaling pathways, including Ca2+ signaling. Phosphatidylserine (PtdSer) mediates various physiological functions; however, junctional PtdSer composition and the role of PtdSer in Ca2+ signaling and Ca2+-dependent gene regulation are not understood. Here, we show that STIM1-formed junctions are required for PI(4)P/PtdSer exchange by ORP5 and ORP8, which have reciprocal lipid exchange modes and function as a rheostat that sets the junctional PtdSer/PI(4)P ratio. Targeting the ORP5 and ORP8 and their lipid transfer ORD domains to PM subdomains revealed that ORP5 sets low and ORP8 high junctional PI(4)P/PtdSer ratio that controls STIM1-STIM1 and STIM1-Orai1 interaction and the activity of the SERCA pump to determine the pattern of receptor-evoked Ca2+ oscillations, and consequently translocation of NFAT to the nucleus. Significantly, targeting the ORP5 and ORP8 ORDs to the STIM1 ER subdomain reversed their function. Notably, changing PI(4)P/PtdSer ratio by hydrolysis of PM or ER PtdSer with targeted PtdSer-specific PLA1a1 reproduced the ORPs function. The function of the ORPs is determined both by their differential lipid exchange modes and by privileged localization at the ER/PM subdomains. These findings reveal a role of PtdSer as a signaling lipid that controls the available PM PI(4)P, the unappreciated role of ER PtdSer in cell function, and the diversity of the ER/PM junctions. The effect of PtdSer on the junctional PI(4)P level should have multiple implications in cellular signaling and functions.

TRPC3 channel gating by lipids requires localization at the ER/PM junctions defined by STIM1.

TRPC3, a member of the transient receptor potential (TRP) superfamily of cation channels, is a lipid-regulated, Ca2+-permeable channel that mediates essential components of the receptor evoked Ca2+ signal. The modes and mechanisms by which lipids regulate TRPC3 and other members of the TRPC channel family are not well understood. Here, we report that PI(4,5)P2 regulates TRPC3 in three independent modes. PLC-dependent hydrolysis generates diacylglycerol (DAG) that interacts with lipid-binding site 2 in the channel pore. PI(4,5)P2 interacts with lipid site 1 to inhibit TRPC3 opening and regulate access of DAG to the pore lipid site 2. PI(4,5)P2 is required for regulating pore ionic selectivity by receptor stimulation. Notably, the activation and regulation of TRPC3 by PI(4,5)P2 require recruitment of TRPC3 to the ER/PM junctions at a PI(4,5)P2-rich domain. Accordingly, we identified an FFAT site at the TRPC3 N-terminal loop within the linker helices that envelope the C-terminus pole helix. The FFAT site interacts with the ER-resident VAPB to recruit TRPC3 to the ER/PM junctions and control its receptor-mediated activation. The TRPC3's lipid interacting sites are fully conserved in TRPC6 and TRPC7 and in part in other TRPC channels. These findings inform on multiple modes of regulation of ion channels by lipids that may be relevant to diseases affected by aberrant TRPC channel functions.

Ca2+ Signaling in Exocrine Cells.

Calcium (Ca2+) and cyclic AMP (cAMP) signaling cross talk and synergize to stimulate the cardinal functions of exocrine cells, regulated exocytosis, and fluid and electrolyte secretion. This physiological process requires the organization of the two signaling pathways into complexes at defined cellular domains and close placement. Such domains are formed by membrane contact sites (MCS). This review discusses the basic properties of Ca2+ signaling in exocrine cells, the role of MCS in the organization of cell signaling and in cross talk and synergism between the Ca2+ and cAMP signaling pathways and, finally, the mechanism by which the Ca2+ and cAMP pathways synergize to stimulate epithelial fluid and electrolyte secretion.

Ca2+ Influx Channel Inhibitor SARAF Protects Mice From Acute Pancreatitis.

BACKGROUND & AIMS: Pancreatitis is characterized by increased influx of Ca2+ into acinar cells, by unknown mechanisms. Inhibitors of Ca2+ influx channels could be effective in treating acute pancreatitis, but these have deleterious side effects that can result in death. We investigated the expression patterns and functions of acinar cell Ca2+ channels and factors that regulate them during development of acute pancreatitis, along with changes in the channel inactivator store-operated calcium entry-associated regulatory factor (SARAF). We investigated whether SARAF is a target for treatment of acute pancreatitis and its status in human with pancreatitis. METHODS: We generated mice that expressed SARAF tagged with hemagglutinin, using CRISPR/Cas9 gene editing, and isolated acinar cells. We also performed studies with Saraf-/- mice, Sarafzf/zf mice, mice without disruption of Saraf (control mice), and mice that overexpress fluorescently labeled SARAF in acinar cells. We analyzed interactions between stromal interaction molecule 1 (STIM1) and SARAF in HEK cells stimulated with carbachol using fluorescence resonance energy transfer microscopy and immunoprecipitation. Mice were given injections of caerulein or L-arginine to induce pancreatitis. Pancreatic tissues and blood samples were collected and levels of serum amylase, trypsin, tissue damage, inflammatory mediators, and inflammatory cells were measured. We performed quantitative polymerase chain reaction analyses of pancreatic tissues from 6 organ donors without pancreatic disease (controls) and 8 patients with alcohol-associated pancreatitis. RESULTS: Pancreatic levels of Ca2+ influx channels or STIM1 did not differ significantly between acinar cells from mice with vs. without pancreatitis. By contrast, pancreatic levels of Saraf messenger RNA and SARAF protein initially markedly increased but then decreased during cell stimulation or injection of mice with caerulein, resulting in excessive Ca2+ influx. STIM1 interacted stably with SARAF following stimulation of HEK or mouse acinar cells with physiologic levels of carbachol, but only transiently following stimulation with pathologic levels of carbachol, leading to excessive Ca2+ influx. We observed reduced levels of SARAF messenger RNA in pancreatic tissues from patients with pancreatitis, compared with controls. SARAF knockout mice developed more severe pancreatitis than control mice after administration of caerulein or L-arginine, and pancreatic acinar cells from these mice had significant increases in Ca2+ influx. Conversely, overexpression of SARAF in acini reduced Ca2+ influx, eliminated inflammation, and reduced severity of acute pancreatitis. CONCLUSIONS: In mice with pancreatitis, SARAF initially increases but is then degraded, resulting in excessive, pathological Ca2+ influx by acinar cells. SARAF knockout mice develop more severe pancreatitis than control mice, whereas mice that express SARAF from a transgene in acinar cells develop less-severe pancreatitis. SARAF therefore appears to prevent pancreatic damage during development of acute pancreatitis. Strategies to stabilize or restore SARAF to acinar cells might be developed for treatment of pancreatitis.

Anoctamin 8 tethers endoplasmic reticulum and plasma membrane for assembly of Ca2+ signaling complexes at the ER/PM compartment.

Communication and material transfer between membranes and organelles take place at membrane contact sites (MCSs). MCSs between the ER and PM, the ER/PM junctions, are the sites where the ER Ca2+ sensor STIM1 and the PM Ca2+ influx channel Orai1 cluster. MCSs are formed by tether proteins that bridge the opposing membranes, but the identity and role of these tethers in receptor-evoked Ca2+ signaling is not well understood. Here, we identified Anoctamin 8 (ANO8) as a key tether in the formation of the ER/PM junctions that is essential for STIM1-STIM1 interaction and STIM1-Orai1 interaction and channel activation at a ER/PM PI(4,5)P2-rich compartment. Moreover, ANO8 assembles all core Ca2+ signaling proteins: Orai1, PMCA, STIM1, IP3 receptors, and SERCA2 at the ER/PM junctions to mediate a novel form of Orai1 channel inactivation by markedly facilitating SERCA2-mediated Ca2+ influx into the ER. This controls the efficiency of receptor-stimulated Ca2+ signaling, Ca2+ oscillations, and duration of Orai1 activity to prevent Ca2+ toxicity. These findings reveal the central role of MCSs in determining efficiency and fidelity of cell signaling.

CRAC channels in secretory epithelial cell function and disease.

The receptor-evoked Ca2+ signal in secretory epithelia mediate many cellular functions essential for cell survival and their most fundamental functions of secretory granules exocytosis and fluid and electrolyte secretion. Ca2+ influx is a key component of the receptor-evoked Ca2+ signal in secretory cell and is mediated by both TRPC and the STIM1-activated Orai1 channels that mediates the Ca2+ release-activated current (CRAC) Icrac. The core components of the receptor-evoked Ca2+ signal are assembled at the ER/PM junctions where exchange of materials between the plasma membrane and internal organelles take place, including transfer of lipids and Ca2+. The Ca2+ signal generated at the confined space of the ER/PM junctions is necessary for activation of the Ca2+-regulated proteins and ion channels that mediate exocytosis with high fidelity and tight control. In this review we discuss the general properties of Ca2+ signaling, PI(4,5)P2 and other lipids at the ER/PM junctions with regard to secretory cells function and disease caused by uncontrolled Ca2+ influx.

Lipids at membrane contact sites: cell signaling and ion transport.

Communication between organelles is essential to coordinate cellular functions and the cell's response to physiological and pathological stimuli. Organellar communication occurs at membrane contact sites (MCSs), where the endoplasmic reticulum (ER) membrane is tethered to cellular organelle membranes by specific tether proteins and where lipid transfer proteins and cell signaling proteins are located. MCSs have many cellular functions and are the sites of lipid and ion transfer between organelles and generation of second messengers. This review discusses several aspects of MCSs in the context of lipid transfer, formation of lipid domains, generation of Ca2+ and cAMP second messengers, and regulation of ion transporters by lipids.

Restoration of CFTR Activity in Ducts Rescues Acinar Cell Function and Reduces Inflammation in Pancreatic and Salivary Glands of Mice.

BACKGROUND & AIMS: Sjögren's syndrome and autoimmune pancreatitis are disorders with decreased function of salivary, lacrimal glands, and the exocrine pancreas. Nonobese diabetic/ShiLTJ mice and mice transduced with the cytokine BMP6 develop Sjögren's syndrome and chronic pancreatitis and MRL/Mp mice are models of autoimmune pancreatitis. Cystic fibrosis transmembrane conductance regulator (CFTR) is a ductal Cl- channel essential for ductal fluid and HCO3- secretion. We used these models to ask the following questions: is CFTR expression altered in these diseases, does correction of CFTR correct gland function, and most notably, does correcting ductal function correct acinar function? METHODS: We treated the mice models with the CFTR corrector C18 and the potentiator VX770. Glandular, ductal, and acinar cells damage, infiltration, immune cells and function were measured in vivo and in isolated duct/acini. RESULTS: In the disease models, CFTR expression is markedly reduced. The salivary glands and pancreas are inflamed with increased fibrosis and tissue damage. Treatment with VX770 and, in particular, C18 restored salivation, rescued CFTR expression and localization, and nearly eliminated the inflammation and tissue damage. Transgenic overexpression of CFTR exclusively in the duct had similar effects. Most notably, the markedly reduced acinar cell Ca2+ signaling, Orai1, inositol triphosphate receptors, Aquaporin 5 expression, and fluid secretion were restored by rescuing ductal CFTR. CONCLUSIONS: Our findings reveal that correcting ductal function is sufficient to rescue acinar cell function and suggests that CFTR correctors are strong candidates for the treatment of Sjögren's syndrome and pancreatitis.

Ca2+ influx at the ER/PM junctions.

Ca2+ influx across the plasma membrane is a key component of the receptor-evoked Ca2+ signaling that mediate numerous cell functions and reload the ER after partial or full ER Ca2+ store depletion. Ca2+ influx is activated in response to Ca2+ release from the ER, a concept developed by Jim Putney, and the channels mediating the influx are thus called store-operated Ca2+ influx channels, or SOCs. The molecular identity of the SOCs has been determined with the identification of the TRPC channels, STIM1 and the Orai channels. These channels are targeted to, operate and are regulated when at the ER/PM junctions. ER/PM junctions are a form of membrane contact sites (MCSs) that are present in all parts of the cells, where the ER makes contacts with cellular membranes and organelles. MCSs have many cellular functions, and are the sites of lipid and Ca2+ transport and delivery between organelles. This short review discusses aspects of MCSs in the context of Ca2+ transport.

Orai1-Mediated Antimicrobial Secretion from Pancreatic Acini Shapes the Gut Microbiome and Regulates Gut Innate Immunity.

The gut microbiome participates in numerous physiologic functions and communicates intimately with the host immune system. Antimicrobial peptides are critical components of intestinal innate immunity. We report a prominent role for antimicrobials secreted by pancreatic acini in shaping the gut microbiome that is essential for intestinal innate immunity, barrier function, and survival. Deletion of the Ca2+ channel Orai1 in pancreatic acini of adult mice resulted in 60%-70% mortality within 3 weeks. Despite robust activation of the intestinal innate immune response, mice lacking acinar Orai1 exhibited intestinal bacterial outgrowth and dysbiosis, ultimately causing systemic translocation, inflammation, and death. While digestive enzyme supplementation was ineffective, treatments constraining bacterial outgrowth (purified liquid diet, broad-spectrum antibiotics) rescued survival, feeding, and weight gain. Pancreatic levels of cathelicidin-related antimicrobial peptide (CRAMP) were reduced, and supplement of synthetic CRAMP prevented intestinal disease. These findings reveal a critical role for antimicrobial pancreatic secretion in gut innate immunity.

The TRPCs, Orais and STIMs in ER/PM Junctions.

The Ca(2+) second messenger is initiated at ER/PM junctions and propagates into the cell interior to convey the receptor information. The signal is maintained by Ca(2+) influx across the plasma membrane through the Orai and TRPC channels. These Ca(2+) influx channels form complexes at ER/PM junctions with the ER Ca(2+) sensor STIM1, which activates the channels. The function of STIM1 is modulated by other STIM isoforms like STIM1L, STIM2 and STIM2.1/STIM2ß and by SARAF, which mediates the Ca(2+)-dependent inhibition of Orai channels. The ER/PM junctions are formed at membrane contact sites by tethering proteins that generate several types of ER/PM junctions, such as PI(4,5)P2-poor and PI(4,5)P2-rich domains. This chapter discusses several properties of the TRPC channels, the Orai channels and the STIMs, their key interacting proteins and how interaction of the STIMs with the channels gates their activity. The chapter closes by highlighting open questions and potential future directions in this field.

Fusion of lysosomes with secretory organelles leads to uncontrolled exocytosis in the lysosomal storage disease mucolipidosis type IV.

Mutations in TRPML1 cause the lysosomal storage disease mucolipidosis type IV (MLIV). The role of TRPML1 in cell function and how the mutations cause the disease are not well understood. Most studies focus on the role of TRPML1 in constitutive membrane trafficking to and from the lysosomes. However, this cannot explain impaired neuromuscular and secretory cells' functions that mediate regulated exocytosis. Here, we analyzed several forms of regulated exocytosis in a mouse model of MLIV and, opposite to expectations, we found enhanced exocytosis in secretory glands due to enlargement of secretory granules in part due to fusion with lysosomes. Preliminary exploration of synaptic vesicle size, spontaneous mEPSCs, and glutamate secretion in neurons provided further evidence for enhanced exocytosis that was rescued by re-expression of TRPML1 in neurons. These features were not observed in Niemann-Pick type C1. These findings suggest that TRPML1 may guard against pathological fusion of lysosomes with secretory organelles and suggest a new approach toward developing treatment for MLIV.

The ER/PM microdomain, PI(4,5)P2 and the regulation of STIM1-Orai1 channel function.

All forms of cell signaling occur in discreet cellular microdomains in which the ER is the main participant and include microdomains formed by the ER with lysosomes, endosomes, the nucleus, mitochondria and the plasma membrane. In the microdomains the two opposing organelles transfer and exchange constituents including lipids and ions. As is the case for other forms of signaling pathways, many components of the receptor-evoked Ca(2+) signal are clustered at the ER/PM microdomain, including the Orai1-STIM1 complex. This review discusses recent advances in understanding the molecular components that tether the ER and plasma membrane to form the ER/PM microdomains in which PI(4,5)P2 is enriched, and how dynamic targeting of the Orai1-STIM1 complex to PI(4,5)P2-poor and PI(4,5)P2-rich microdomains controls the activity of Orai1 and its regulation by Ca(2+) that is mediated by SARAF.

Translocation between PI(4,5)P2-poor and PI(4,5)P2-rich microdomains during store depletion determines STIM1 conformation and Orai1 gating.

The Orai1-STIM1 current undergoes slow Ca(2+)-dependent inactivation (SCDI) mediated by the binding of SARAF to STIM1. Here we report the use of SCDI by SARAF as a probe of the conformation and microdomain localization of the Orai1-STIM1 complex. We find that the interaction of STIM1 with Orai1 carboxyl terminus (C terminus) and the STIM1 K-domain are required for the interaction of SARAF with STIM1 and SCDI. STIM1-Orai1 must be in a PM/ER microdomain tethered by E-Syt1, stabilized by septin4 and enriched in PI(4,5)P2 for STIM1-SARAF interaction. Targeting STIM1 to PI(4,5)P2-rich and -poor microdomains reveals that SARAF-dependent SCDI is observed only when STIM1-Orai1 are within the PI(4,5)P2-rich microdomain. Notably, store depletion results in transient localization of STIM1-Orai1 in the PI(4,5)P2-poor microdomain, which then translocates to the PI(4,5)P2-rich domain. These findings reveal the role of PM/ER tethers in the regulation of Orai1 function and a mode of regulation by PI(4,5)P2 involving translocation between PI(4,5)P2 microdomains.

The TRPCs-STIM1-Orai interaction.

Ca(2+) signaling entails receptor-stimulated Ca(2+) release from the ER stores that serves as a signal to activate Ca(2+) influx channels present at the plasma membrane, the store-operated Ca(2+) channels (SOCs). The two known SOCs are the Orai and TRPC channels. The SOC-dependent Ca(2+) influx mediates and sustains virtually all Ca(2+)-dependent regulatory functions. The signal that transmits the Ca(2+) content of the ER stores to the plasma membrane is the ER resident, Ca(2+)-binding protein STIM1. STIM1 is a multidomain protein that clusters and dimerizes in response to Ca(2+) store depletion leading to activation of Orai and TRPC channels. Activation of the Orais by STIM1 is obligatory for their function as SOCs, while TRPC channels can function as both STIM1-dependent and STIM1-independent channels. Here we discuss the different mechanisms by which STIM1 activates the Orai and TRPC channels, the emerging specific and non-overlapping physiological functions of Ca(2+) influx mediated by the two channel types, and argue that the TRPC channels should be the preferred therapeutic target to control the toxic effect of excess Ca(2+) influx.

cAMP and Ca²âº signaling in secretory epithelia: crosstalk and synergism.

The Ca(2+) and cAMP/PKA pathways are the primary signaling systems in secretory epithelia that control virtually all secretory gland functions. Interaction and crosstalk in Ca(2+) and cAMP signaling occur at multiple levels to control and tune the activity of each other. Physiologically, Ca(2+) and cAMP signaling operate at 5-10% of maximal strength, but synergize to generate the maximal response. Although synergistic action of the Ca(2+) and cAMP signaling is the common mode of signaling and has been known for many years, we know very little of the molecular mechanism and mediators of the synergism. In this review, we discuss crosstalk between the Ca(2+) and cAMP signaling and the function of IRBIT (IP3 receptors binding protein release with IP3) as a third messenger that mediates the synergistic action of the Ca(2+) and cAMP signaling.

The gatekeepers of mitochondrial calcium influx: MICU1 and MICU2.

The receptor-evoked Ca(2+) signal is sensed and translated by mitochondria. Physiological cytoplasmic Ca(2+) ([Ca(2+)]c) oscillations result in mitochondrial Ca(2+) ([Ca(2+)]m) oscillations, while large and sustained [Ca(2+)]c increase results in a pathologic increase in basal [Ca(2+)]m and in Ca(2+) accumulation. The physiological [Ca(2+)]m signal regulates [Ca(2+)]c and stimulates oxidative metabolism, while excess Ca(2+) accumulation causes cell stress leading to cell death. [Ca(2+)]m is determined by Ca(2+) uptake mediated by the mitochondria Ca(2+) uniporter (MCU) channel and by Na(+)- and H(+)-coupled Ca(2+) extrusion.

Convergent regulation of the lysosomal two-pore channel-2 by Mg²âº, NAADP, PI(3,5)P2 and multiple protein kinases.

Lysosomal Ca(2+) homeostasis is implicated in disease and controls many lysosomal functions. A key in understanding lysosomal Ca(2+) signaling was the discovery of the two-pore channels (TPCs) and their potential activation by NAADP. Recent work concluded that the TPCs function as a PI(3,5)P2 activated channels regulated by mTORC1, but not by NAADP. Here, we identified Mg(2+) and the MAPKs, JNK and P38 as novel regulators of TPC2. Cytoplasmic Mg(2+) specifically inhibited TPC2 outward current, whereas lysosomal Mg(2+) partially inhibited both outward and inward currents in a lysosomal lumen pH-dependent manner. Under controlled Mg(2+), TPC2 is readily activated by NAADP with channel properties identical to those in response to PI(3,5)P2. Moreover, TPC2 is robustly regulated by P38 and JNK. Notably, NAADP-mediated Ca(2+) release in intact cells is regulated by Mg(2+), PI(3,5)P2, and P38/JNK kinases, thus paralleling regulation of TPC2 currents. Our data affirm a key role for TPC2 in NAADP-mediated Ca(2+) signaling and link this pathway to Mg(2+) homeostasis and MAP kinases, pointing to roles for lysosomal Ca(2+) in cell growth, inflammation and cancer.

Molecular determinants mediating gating of Transient Receptor Potential Canonical (TRPC) channels by stromal interaction molecule 1 (STIM1).

Transient receptor potential canonical (TRPC) channels mediate a critical part of the receptor-evoked Ca(2+) influx. TRPCs are gated open by the endoplasmic reticulum Ca(2+) sensor STIM1. Here we asked which stromal interaction molecule 1 (STIM1) and TRPC domains mediate the interaction between them and how this interaction is used to open the channels. We report that the STIM1 Orai1-activating region domain of STIM1 interacts with the TRPC channel coiled coil domains (CCDs) and that this interaction is essential for opening the channels by STIM1. Thus, disruption of the N-terminal (NT) CCDs by triple mutations eliminated TRPC surface localization and reduced binding of STIM1 to TRPC1 and TRPC5 while increasing binding to TRPC3 and TRPC6. Single mutations in TRPC1 NT or C-terminal (CT) CCDs reduced interaction and activation of TRPC1 by STIM1. Remarkably, single mutations in the TRPC3 NT CCD enhanced interaction and regulation by STIM1. Disruption in the TRPC3 CT CCD eliminated regulation by STIM1 and the enhanced interaction caused by NT CCD mutations. The NT CCD mutations converted TRPC3 from a TRPC1-dependent to a TRPC1-independent, STIM1-regulated channel. TRPC1 reduced the FRET between BFP-TRPC3 and TRPC3-YFP and between CFP-TRPC3-YFP upon stimulation. Accordingly, knockdown of TRPC1 made TRPC3 STIM1-independent. STIM1 dependence of TRPC3 was reconstituted by the TRPC1 CT CCD alone. Knockout of Trpc1 and Trpc3 similarly inhibited Ca(2+) influx, and inhibition of Trpc3 had no further effect on Ca(2+) influx in Trpc1(-/-) cells. Cell stimulation enhanced the formation of Trpc1-Stim1-Trpc3 complexes. These findings support a model in which the TRPC3 NT and CT CCDs interact to shield the CT CCD from interaction with STIM1. The TRPC1 CT CCD dissociates this interaction to allow the STIM1 Orai1-activating region within STIM1 access to the TRPC3 CT CCD and regulation of TRPC3 by STIM1. These studies provide evidence that the TRPC channel CCDs participate in channel gating.