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 Ca signaling. Phosphatidylserine (PtdSer) mediates various physiological functions; however, junctional PtdSer composition and the role of PtdSer in Ca signaling and Ca-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.

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.

Ca Signaling in Exocrine Cells.

Calcium (Ca) 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).

Ca Influx Channel Inhibitor SARAF Protects Mice From Acute Pancreatitis.

Background & Aims: Pancreatitis is characterized by increased influx of Ca into acinar cells, by unknown mechanisms. Inhibitors of Ca 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 Ca 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).

Anoctamin 8 tethers endoplasmic reticulum and plasma membrane for assembly of Ca 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 Ca sensor STIM1 and the PM Ca 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 Ca signaling is not well understood.

CRAC channels in secretory epithelial cell function and disease.

The receptor-evoked Ca 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. Ca influx is a key component of the receptor-evoked Ca signal in secretory cell and is mediated by both TRPC and the STIM1-activated Orai1 channels that mediates the Ca release-activated current (CRAC) I. The core components of the receptor-evoked Ca 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 Ca.

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.

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 HCO secretion.

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.

Ca influx at the ER/PM junctions.

Ca influx across the plasma membrane is a key component of the receptor-evoked Ca signaling that mediate numerous cell functions and reload the ER after partial or full ER Ca store depletion. Ca influx is activated in response to Ca release from the ER, a concept developed by Jim Putney, and the channels mediating the influx are thus called store-operated Ca 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.

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.

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.

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.

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.

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.

cAMP and Ca²⁺ 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.

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.

Convergent regulation of the lysosomal two-pore channel-2 by Mg²⁺, 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.

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.

The STIM1 CTID domain determines access of SARAF to SOAR to regulate Orai1 channel function.

Ca(2+) influx by store-operated Ca(2+) channels (SOCs) mediates all Ca(2+)-dependent cell functions, but excess Ca(2+) influx is highly toxic. The molecular components of SOC are the pore-forming Orai1 channel and the endoplasmic reticulum Ca(2+) sensor STIM1. Slow Ca(2+)-dependent inactivation (SCDI) of Orai1 guards against cell damage, but its molecular mechanism is unknown.

The intracellular Ca²⁺ channels of membrane traffic.

Regulation of organellar fusion and fission by Ca ( 2+) has emerged as a central paradigm in intracellular membrane traffic. Originally formulated for Ca ( 2+) -driven SNARE-mediated exocytosis in the presynaptic terminals, it was later expanded to explain membrane traffic in other exocytic events within the endo-lysosomal system. The list of processes and conditions that depend on the intracellular membrane traffic includes aging, antigen and lipid processing, growth factor signaling and enzyme secretion.

Membrane potential regulates nicotinic acid adenine dinucleotide phosphate (NAADP) dependence of the pH- and Ca2+-sensitive organellar two-pore channel TPC1.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent second messenger that mobilizes Ca(2+) from the acidic endolysosomes by activation of the two-pore channels TPC1 and TPC2. The channel properties of human TPC1 have not been studied before, and its cellular function is not known. In the present study, we characterized TPC1 incorporated into lipid bilayers.