Orai1 and STIM1 in ER/PM junctions: roles in pancreatic cell function and dysfunction

Abstract

Membrane contact sites (MCS) are critical junctions that form between the endoplasmic reticulum (ER) and membranes of various organelles, including the plasma membrane (PM). Signaling complexes, including mediators of Ca²⁺ signaling, are assembled within MCS, such as the ER/PM junction. This is most evident in polarized epithelial cells, such as pancreatic cells. Core Ca²⁺ signaling proteins cluster at the apical pole, the site of inositol 1,4,5-trisphosphate-mediated Ca²⁺ release and Orai1/transient receptor potential canonical-mediated store-dependent Ca²⁺ entry. Recent advances have characterized the proteins that tether the membranes at MCS and the role of these proteins in modulating physiological and pathological intracellular signaling. This review discusses recent advances in the characterization of Ca²⁺ signaling at ER/PM junctions and the relation of these junctions to physiological and pathological Ca²⁺ signaling in pancreatic acini.

intracellular signaling pathways are mediated by signaling complexes that interact with each other, often synergistically, to enhance downstream physiological responses. These signal transduction proteins must be in close physical proximity to allow these interactions. Each pathway is composed of several proteins with defined functions that regulate both the spatial and temporal fidelity of the signals and convey them to targets within the cell interior. The proteins that make the pathways are assembled into signaling complexes that interact with each other in molecular distances. To promote organized signaling cascades, the various steps are compartmentalized within specific cellular sites. Compartmentalized signaling complexes are present in the plasma membrane (PM), endosomes, lysosomes, mitochondria, and the endoplasmic reticulum (ER). Within these compartments, signaling complexes are further restricted to defined microdomains.

In recent years, an improved molecular understanding of these microdomains has emerged, especially of the membrane contact sites (MCS) that form between the ER and other cellular membranes. MCS are formed by tethering proteins at the ER/PM junctions (7, 31), ER/mitochondria (113), ER/endosomes/lysosomes (81) [vacuoles in yeast (85)], and ER/nuclear membrane (85). Many MCS serve to communicate and transfer material between membranes, particularly lipids in a nonvesicular form of transport (50). This review focuses on one particular MCS, the ER/PM junction, due to its role in Orai1/stromal interacting molecule 1 (STIM1)-mediated Ca²⁺ signaling. Discussion of other MCS can be found in several excellent recent reviews (31, 50, 81, 85, 88, 113).

The Pancreatic ER/PM Junctions

Secretory epithelial cells, including those in the pancreas, lung, salivary glands, and other gastrointestinal organs are all polarized structurally with basal and apical poles. This structure allows their polarized functions of exocytosis and transcellular fluid and electrolyte secretion (54). Accordingly, signaling machinery in these cell types, including the Ca²⁺ signaling apparatus, are organized into complexes in ER/PM junctions. The ER/PM junctions in secretory cells are most prominent at the apical pole. All G protein-coupled receptors (GPCRs) examined, including the cholecystokinin and muscarinic receptors that signal through changes in cytoplasmic Ca²⁺ {intracellular Ca²⁺ concentration ([Ca²⁺]_i)} and the vasoactive intestinal peptide receptors that signal by increasing cellular cAMP, are localized at the tight junctions (32, 99). Immunolocalization and functional studies show that all inositol 1,4,5-trisphosphate (IP₃) receptors (IP₃Rs) are expressed at high levels at the apical pole (56, 126), with IP₃R2 and IP₃R3 being the dominant IP₃Rs in exocrine secretory cells (125). The PM Ca²⁺-ATPase pump (PMCA) isoform 4 is confined to the apical pole (99), while the housekeeping PMCA1 is localized at the lateral and basal membranes (55). Exocrine cells also express high level of transient receptor potential canonical 1 (TRPC1), TRPC3, and TRPC6 at their apical pole (34, 43). The store-operated Orai1 channel is markedly enriched at the apical pole, and, on depletion of ER Ca²⁺, the ER Ca²⁺ sensor STIM1 clusters at the apical pole (34). Finally, the scaffolding Homer proteins that bind IP₃Rs, TRPC channels, GPCRs (117, 122), and PMCA (121) are confined to the apical pole.

The placement of Ca²⁺ signaling components at ER/PM junctions in exocrine cells accounts for polarized Ca²⁺ signaling in these cells. Polarized cell signaling is designed to meet the specific functions of the cells. This is reflected very well in the function of pancreatic and salivary acinar cells. The cardinal function of these cells is exocytosis of digestive enzymes and secretion of isotonic, NaCl-rich fluid (54). Both secretory functions depend on critical apical pole proteins and transporters. Pancreatic exocytosis is mediated by increases in free cytoplasmic Ca²⁺ ([Ca²⁺]_i) (66), while exocytosis by salivary gland acinar cells is triggered by increases in cytoplasmic cAMP (86). Fluid secretion by both acinar cell types follows Ca²⁺ oscillations at the apical pole. The receptor-evoked Ca²⁺ signal starts with Ca²⁺ release from the ER, followed by activation of Ca²⁺ influx across the PM through the Orai and TRPC store-operated Ca²⁺ influx channels (SOCs). ER Ca²⁺ release is limited, and Ca²⁺ influx sustains the Ca²⁺ signal and reloads intracellular/ER stores with Ca²⁺ at the end of the stimulation period. The increase in [Ca²⁺]_i activates the PMCA and ER/sarcoplasmic reticulum (SR) Ca²⁺-ATPase (SERCA) pump, which remove Ca²⁺ from the cytoplasm to restore basal cytoplasmic Ca²⁺. At physiological stimulus intensity, the channels periodically inactivate, and the cycle of Ca²⁺ release and influx is repeated, resulting in Ca²⁺ oscillations. Overactivation of Ca²⁺ influx causes a sustained, pathological increase in [Ca²⁺]_i. Hence, Ca²⁺ influx mediates both physiological Ca²⁺ oscillations and pathological sustained increases in [Ca²⁺]_i.

An increase in [Ca²⁺]_i activates the Ca²⁺-activated Cl⁻ channel anoctamin 1 (ANO1) [transmembrane protein (TMEM) 16A] at the apical membrane (9, 79) and K⁺ channels at both the apical and basolateral membranes (2, 89, 90). This results in K⁺ and Cl⁻ efflux and cell shrinkage. Electrical neutrality is achieved by transcellular Na⁺ flux through the tight junctions. NaCl secretion dives fluid secretion through the water channel aquaporin 5 (25). Cell shrinkage inhibits Ca²⁺ signaling via unknown mechanisms; this results in [Ca²⁺]_i reduction back to baseline, inhibition of the Cl⁻ and K⁺ channels, and inhibition of fluid secretion. Simultaneously, cell shrinkage activates the basolateral Na⁺-K⁺-2Cl⁻ cotransporter 1 (NKCC1) (54, 65), which is regulated by the cell volume and osmolality sensitive kinases WNK (with no lysine) and SPAK (Ste20-related proline-alanine-rich kinase) (25, 39). The Na⁺ entering the cells through NKCC1 is exchanged with K⁺ by the Na⁺ pump. Restoration of cytoplasmic K⁺ and Cl⁻ by NKCC1 is followed first by restoration of cell volume and then by a second Ca²⁺ spike, initiating another secretory event. In this manner, acinar cells function as fluid and electrolyte secretory pumps.

The discussion above highlights the reason for generation of signaling mediators at the apical pole of epithelial cells. Indeed, the Ca²⁺ signal in acinar (40, 106) and duct cells (56, 99) always initiates at the apical pole and propagates to the basal pole. Moreover, each receptor has a Ca²⁺ signaling signature with a defined initiation site and propagation pattern (99), which is regulated by specific RGS (regulators of G protein signaling) proteins (118, 127). Mapping receptor-stimulated Ca²⁺ signaling by localized stimulation at cellular microdomains revealed much higher sensitivity of the apical than the basal pole to agonist stimulation (57), likely due to the high level of GPCRs at this pole, as described above. Physiological stimulus intensity evokes Ca²⁺ oscillations that can remain confined to the apical pole (82), where they stimulate the Ca²⁺-activated Cl⁻ channels (79). A mitochondrial belt at the apical pole around the secretory granules helps confine the Ca²⁺ signal to the apical pole (5, 107). Similarly, localized release of IP₃ at low concentrations readily releases Ca²⁺ from the ER at the apical pole, while a much higher concentration of IP₃ is needed to release Ca²⁺ from the basal pole (24). Although Ca²⁺ can enter the cells across the basolateral membrane when presented only to this membrane (68), Ca²⁺ mainly enters the cells when Ca²⁺ release from the ER activates the SOC Orai1 and the TRPC channels at the apical pole (13, 34). Localization of the TRPC3 and Orai1 channels allowing Ca²⁺ entry at the ER/PM junctions of acinar cells is illustrated in Fig. 1.

Fig. 1.

Clustering of Ca²⁺ influx channels and Ca²⁺ influx at the apical pole of pancreatic acini. A and B: colocalization of inositol 1,4,5-trisphosphate receptor 3 (IP₃R3) and of transient receptor potential canonical 3 (A) and Orai1 (B) at the apical pole of pancreatic acini. C: polarized pancreatic acinar cells (note the apical granules at the differential interference contrast image, bottom right) was treated with 0.5 mM carbachol and 25 μM of the endoplasmic reticulum (ER)/sarcoplasmic reticulum Ca²⁺-ATPase inhibitor cyclopiazonic acid in Ca²⁺-free solution to deplete the stores and maximally activate Ca²⁺ influx. The cell was then rapidly perfused with a solution containing 5 mM Ca²⁺, and images were collected every 0.3 s to identify the site on Ca²⁺ influx. It is clear that Ca²⁺ enters the cell primarily from the apical region, where the Ca²⁺ influx channels are clustered. A is reproduced from Ref. 43, and B and C are from Ref. 34.

Components of the cAMP-dependent signaling pathway are also localized at the apical pole ER/PM junctions of acinar cells. Several Ca²⁺-dependent adenylyl cyclases (ACs) are localized at the apical pole (73). Localization of ACs is determined by A-kinase anchoring proteins that interact with apical cytoskeletal proteins like Ezrin (95, 128). Clear compartmentalization of all components of the cAMP pathway has been extensively demonstrated in muscles, where they are expressed at the SR/PM junctions (23, 29). Moreover, receptor stimulation causes large elevations in cAMP, leading to PKA activation at the junctions (23, 92). In muscles and secretory cells, the Ca²⁺ and cAMP signaling pathways are in close proximity, allowing their functional interaction. Indeed, cross-activation (15) and synergism (1, 80) between these two pathways are well established. Of particular interest is the regulation of AC8 and perhaps other Ca²⁺-dependent ACs by Ca²⁺ entering the cells through Orai1 (114) and TRPC1 (115). This regulation depends on the presence of all components in caveolae (74) and requires the direct interaction of AC8 with Orai1 (114) and perhaps with TRPC channels. The ER/PM junctions are enriched in caveolae (74).

Several of the key questions in understanding signaling at the ER/PM junctions are how the junctions are formed, how they are regulated, and how they affect cell function. There is very little information on the proteins that form and maintain the junctions in vivo or in any secretory cells. However, studies of nonvesicular lipid transport in yeast (50, 85) and in model mammalian cell systems (for reviews, see Refs. 7, 31, 85) are providing information that is relevant to all cells and is discussed below.

Tethering the ER/PM Junctions

In yeast, ∼40% of the PM is tethered to the ER and is thus a robust system to study the ER/PM junctions. The ER is tethered to the PM by specialized proteins that form and stabilize the ER/PM junctions and regulate their functions. Early work in yeast identified the tricalbins as proteins that localize to the ER/PM junctions and are required for lipid transfer between the membranes (109). Localization of the tricalbins to the junctions requires their interaction with the PM lipids phosphatidylinositol-4-phosphate (PI4P) and phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P₂] (64, 109). Another essential protein for formation of the yeast junctions is Ist2, which has a polybasic domain that interacts with PI(4,5)P₂ (60, 116). Ssy1 is a protein with a similar general domain structure to Ist2 that is targeted to the ER/PM junctions. Ssy1 also has an ER transmembrane domain, a PM lipid-binding domain, and a disordered linker that bridges the ER/PM distance (49). Although the exact function of Ssy1 is not known at present, its localization suggests that it is likely to have a specific role in the ER/PM junction, such as lipid transfer, endocytosis, or signaling. The yeast ER/PM junctions include the vesicle-associated membrane protein-associated proteins (VAPs) Scs2 and Scs22 and their partners, the oxysterol-binding homology (Osh) proteins (103). The exact role of each protein in junction formation is not clear, but deletion of all of them is required to disrupt the yeast ER/PM junction (64).

In the special case of muscle, ER(SR)/PM junction tether proteins have been studied in some details and include the junctophilins (105) and the type 1 ryanodine receptor, which interacts directly with L-type Ca²⁺ channels (3). Junctophilins 3 and 4 may also participate in the ER/PM junctions in nonmyocytes (105), and yeast homologs with similar functions were identified in mammalian cells. The three extended synaptotagmins (E-Syts) (28, 67) are homologs of the yeast tricalbins with multiple Ca²⁺ binding C2 domains and PI(4,5)P₂ binding capability (28). When expressed in mammalian cells, the three E-Syts tether the ER to the PM to form ER/PM junctions (28). However, the E-Syts appear to have distinct functions. E-Syt1, but not E-Syt2 and E-Syt3, affect STIM1-Orai1 function at the ER/PM junctions (7, 62), whereas E-Syt2 and E-Syt3, but not E-Syt1, interact with activated FGF receptors (110) to mediate receptor endocytosis (36). Based on sequence similarity and predicted topology, it is possible that one or more of the 10 ANOs (also known as TMEM16A-J) (8, 94, 120) are homologs of yeast Ist2. VAP-A and VAP-B are homologs of the yeast Scs proteins. In mammalian cells, the VAPs interact with Nir proteins. Nir2 is a lipid transfer and exchange protein that translocates to the PM and maintains the level of PI(4,5)P₂ by exchanging phosphatidic acid with PI(4,5)P₂ (46, 47) during cell stimulation (10, 47). Nir3 maintains PM PI(4,5)P₂ at the resting state (11). The cytoskeletal proteins septin 4 and septin 5 also localize at the junctions and maintain high levels of PI(4,5)P₂ around Orai1 in response to cell stimulation (97). They may also control the size of the ER/PM junctions (12).

Phosphatidylserine (PS) is an important PM lipid that is enriched in the inner leaflet of the PM and functions as a signaling lipid. PS is first synthesized in the ER and is then transferred to the PM. PS can be translocated from the ER to the PM by ANO proteins. ANO6 (TMEM16F) was shown to have a lipid scramblase function that mediates transfer of PS between membrane leaflets (104). The crystal structure of the fungus Nectria haematococca ANO6 homolog suggests a fascinating potential mechanism of PS flipping (6). The transmembrane domains of the Nectria haematococca ANO6 are arranged to form a hydrophilic cavity within the lipid bilayer that can guide the charged group of the phospholipids between the membrane leaflets (6). Whether the mammalian ANO6 handles phospholipids through a similar mechanism remains to be determined.

Proteins that function in controlling PM PS belong to the oxysterol-binding protein-related protein (ORP) family, including Osh3 (108) and Osh6/7 in yeast (69) and ORP5 and ORP8 in mammalian cells (14). In yeast, Osh3 is recruited to the ER/PM junctions, where it interacts with Scs/VAP through its phenylalanines in an acidic tract (FFAT) motif, and with PM PI4P through its pH domain to mediate lipid transfer (103). The structure of Osh4 shows that it can bind PI4P or sterols, suggesting that the Osh/ORP proteins may function to exchange lipids between membranes (16). Such a mechanism was recently shown to be mediated by the ORP5/ORP8 and their yeast homologs (14, 69). Early studies in mammalian cells showed that ORP5 has a cholesterol-binding domain and localizes at the ER/endolysosomal junction by interacting with Niemann Pick type c1 protein to mediate the transfer of cholesterol from late endosomes to the ER (21). Further studies showed that the ORPs, including ORP5, can mediate the transfer of PS from the ER to the PM (61). The mechanism of PS transfer by ORP5 was described recently. When expressed in mammalian cells, ORP5 and ORP8 form ER/PM junctions that require their PI4P binding pH domain (14). ORP5 and ORP8 bind PI4P or PS and exchange lipids between bilayers (14, 69). The coupled countertransport of PI4P from the PM to the ER and of PS from the ER to the PM determines the level of the respective lipids at the respective membranes (14, 69). The significance of this form of lipid transport to Ca²⁺ signaling is not known at present, although it is likely to affect it by determining the PM PS level.

STIM1 and Orai1 at the ER/PM Junctions

The ER Ca²⁺ sensor STIM1 is an ER/PM junctional protein that has a key role in Ca²⁺ signaling and in formation of the ER/PM junctions. STIM1 is a multidomain protein, but only one of the STIM1 domains is requited to activate the SOCs: the Orais and several TRPC channels (13, 51). The NH₂-terminus of STIM1 resides in the ER lumen and has a low Ca²⁺ affinity EF hand and sterile α motif (SAM) domain. The SAM domain aids in STIM1 clustering on depletion of ER Ca²⁺ (100, 102). The EF hand binds Ca²⁺ when the ER is filled to keep STIM1 in an unclustered, close conformation (70, 101). The single transmembrane span is followed by three coiled-coil domains that regulate unfolding of STIM1 (48, 70) to release the STIM1 Orai1 activating region (SOAR) domain (123), also known as CAD (78) and CCb9 (41). SOAR, a 98-residue domain, is the minimal STIM1 domain (123) that dimerizes (119, 123) and interacts with the COOH- and NH₂-terminus of Orai1 (78, 123), leading to full Orai1 activation. SOAR is followed by the COOH-terminal inhibitory domain (CTID) (37), serine/proline rich and polybasic domains (58, 91). Activation of the Orai channels by STIM1 requires Ca²⁺ release from the ER and co-clustering with STIM1 at the ER/PM junctions. At the ER/PM junctions, the STIM1 SOAR domain interacts with coiled-coil domains at the COOH- and NH₂-termini of Orai1, and likely of other Orai channels. In the case of TRPC channels, the SOAR domain interacts with the coiled-coil domain in the COOH-terminus of the channels, either independently (TRPC1) or after dissociation between the channels COOH- and NH₂-termini coiled-coil domains (TRPC3) (51). As will be discussed in the next section, both Orai1 and the TRPC channels participate in mediating physiological and pathological store-dependent Ca²⁺ influx in pancreatic acinar cells.

STIM1 has the canonical domains needed for localization at the ER/PM junctions: an ER localized transmembrane domain, a PI(4,5)P₂ polybasic binding domain, and a coupling domain that spans the ER/PM space. Accordingly, on store depletion, STIM1 clusters and expands the ER/PM junctions (33). Clustering of STIM1 is regulated by the newly discovered STIM1 interacting protein named STIMATE/TMEM110 (38, 87). STIMATE/TMEM110 has ER membrane resident multiple transmembrane domains and a sort cytoplasmic domain that includes a polybasic sequence. STIMATE/TMEM110 interacts with STIM1 to promote its active conformation and stabilizes the STIM1-formed ER/PM junctions (38, 87). Deletion of STIMATE/TMEM110 prevents STIM1 clustering and STIM1-Orai1 interaction to inhibit the Orai1 current and Ca²⁺ influx (38, 87). Another protein that regulates STIM1 function is SARAF (75). SARAF is an ER resident, single transmembrane span protein with a long cytoplasmic domain that mediates the slow Ca²⁺-dependent inactivation of Orai1. Orai1 undergoes two types of Ca²⁺-dependent inactivation, fast inactivation with a time constant of ∼10 ms and slow inactivation with a time constant of 1–2 min (77). A negatively charged STIM1 sequence within CTID (37) is required for Orai1 inactivation (17, 53, 71). CTID regulates the access of SARAF to the STIM1 SOAR domain (37). Notably, SARAF interacts with STIM1 only when STIM1 is at the PI(4,5)P₂-rich ER/PM junctions (62). Interestingly, the interaction between STIMATE/TMEM110 and STIM1 is Orai1 independent and is concurrent with or precedes STIM1-Orai1 interaction (38, 87). Conversely, interaction of SARAF with STIM1 takes place after and indeed requires formation of the STIM1-Orai1 complex (37). Hence, it is possible that, at the resting state, interaction of STIMATE/TMEM110 and SARAF with STIM1 is minimal. Upon store depletion, STIMATE/TMEM110 facilitates STIM1 clustering and stabilizes the active STIM1 conformation at the PI(4,5)P₂-poor domain to fully activate Ca²⁺ influx. When the STIM1-Orai1 complex moves to the PI(4,5)P₂-rich domain, SARAF is recruited to the complex to partially inhibit Ca²⁺ influx. This scenario of early interaction of STIMATE with STIM1 to facilitate STIM1 clustering, and delayed interaction of STIM1 with SARAF to inhibit the channel at the ER/PM junctions PI(4,5)P₂-poor and PI(4,5)P₂-rich domains, is illustrated in Fig. 2, top. Excessive pathological Ca²⁺ influx can occur when the STIMATE or SARAF do not function properly, or when translocation of Orai1-STIM1 to the PI(4,5)P₂-rich domain fails. The Orai1-STIM1 complex may fail to translocate to the PI(4,5)P₂-rich domain, either because the PI(4,5)P₂ at the ER/PM junctions is completely hydrolyzed by strong receptor stimulation, or because STIMATE fails to facilitate STIM1 clustering and translocation, preventing SARAF from interacting with STIM1. Mutation or posttranslational modification of SARAF may also prevent SARAF interaction with STIM1. Loss of SARAF-STIM1 interaction will maintain the Ca²⁺ influx channels fully active, resulting in a sustained pathological increase in [Ca²⁺]_i, as illustrated in Fig. 2, bottom.

Fig. 2.

Ca²⁺ signaling at the ER/plasma membrane (PM) junctions. The ER/PM junctions are tethered by the tethering proteins extended synaptotagmin 1 and an anoctamin isoform (homologs of yeast Ist2) to form a phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P₂]-rich domain at the PM. Physiology: In the resting state, stromal interacting molecule 1 (STIM1) is in an inactive, not clustered, state and does not interact with either SARAF or STIMATE. Orai1 is at the PM PI(4,5)P₂-poor domain and is not clustered. Store depletion recruits STIMATE to STIM1 to facilitate STIM1 clustering and interaction with Orai1 at the PI(4,5)P₂-poor domain to fully activate Orai1 and Ca²⁺ influx. The STIM1-Orai1 complex then translocates to the PI(4,5)P₂-rich ER/PM junctions, where SARAF interacts with STIM1 to mediate Ca²⁺-dependent inactivation of Orai1 and limit Ca²⁺ influx to that required to sustain the physiological Ca²⁺ response. Pathology: When the Orai1-STIM1 complex fails to translocate to the PI(4,5)P₂-rich ER/PM junctions and/or SARAF fails to interact with STIM1 at the PI(4,5)P₂-rich ER/PM junctions to inhibit Ca²⁺ influx, Ca²⁺ influx remains fully activated, resulting in a sustained increase in cytoplasmic Ca²⁺ concentration and cell toxicity, as occurs in acute pancreatitis. SMP, synaptotagmin-like-mitochondrial-lipid binding protein; PB, polybasic.

The Pancreatic ER/PM Junctions in Pathology

In all cell types, excessive and sustained increase in cytoplasmic Ca²⁺ ([Ca²⁺]_i) is highly toxic, leading to cell death by disrupting key cellular functions, including mitochondrial function (20, 84), apoptosis (19), gene regulation (76) and membrane transport (54, 63). The major mechanism underlying excessive increase in [Ca²⁺]_i is overactivation of the various Ca²⁺ influx channels. In nonexcitable cells, the main Ca²⁺ influx channels are the TRPC channels and Orai1 (7, 13). It is well established that the major cause of various pathologies in secretory epithelial cells, including the pancreas, is excessive Ca²⁺ influx (22, 26, 30, 35). Acute pancreatitis is a life-threatening disease with multiple potential etiologies and no cure. Uncontrolled activation of Ca²⁺ influx was shown as the nodal point in triggering the common forms of acute pancreatitis that are initiated by intense receptor stimulation, bile acids, and excessive alcohol consumption (26, 63). Moreover, in vitro experiments showed that buffering [Ca²⁺]_i to prevent the sustained increase in [Ca²⁺]_i protects against all forms of acute pancreatitis (42, 83, 111).

Due to the central role of excessive Ca²⁺ influx in pancreatitis, inhibition of Ca²⁺ influx channels has been considered as a potential treatment for the disease. In principal, inhibitors of TRPC channels, of Orai1, or STIM1 should be beneficial in the treatment of pancreatitis, each of which has advantages and drawbacks. The main advantage of TRPC channel inhibition is that inhibitors should have modest side effects: knockout of TRPC1 (59), TRPC3 (44), or TRPC6 (18) and combinations of these channels (72, 96) all have minor phenotypes. Another advantage is that, when multiple TRPC channels are expressed in the same cells, they heteromultimerize (51, 52, 124), and inhibition of one isoform is sufficient to inhibit all TRPC channel-mediated Ca²⁺ influx, as was found in pancreatic acinar cells (51). Accordingly, knockout of TRPC3 in mice ameliorates the damage observed in acute pancreatitis (44). Similar protection has been observed by inhibition of TRPC3 with pyrazol 3 (45). The disadvantage of inhibiting TRPC3, and possibly other TRPC channels, is that experimental inhibition protects only against mild forms of pancreatitis (44, 45), and the currently available drug is not able to inhibit the fully activated channel (45). Hence more potent TRPC channel inhibitors would be desirable for pancreatitis therapy.

Inhibitors of Orai1 have been tested recently as potential treatments for pancreatitis (27, 112). In vitro studies showed that inhibition of Orai1 prevents the sustained Ca²⁺ increase caused by intense receptor stimulation, by fatty acids ethyl esters (products of alcohol metabolism), and by activation of intracellular trypsin (27). The same and an additional Orai1 inhibitor were used in mice before induction of acute pancreatitis by bile acid, by stimulation with cholecystokinin, or by exposure to alcohol + fatty acids ethyl esters (112). Orai1 blockers partially but significantly protected against all models of acute pancreatitis (112). One significant advantage of Orai1 blockade is that all forms of acute pancreatitis are associated with severe inflammation (93), and Orai1-mediated Ca²⁺ influx is critical for activation of various inflammatory cells (98). Inhibition of Orai1 can, therefore, protect the pancreas by two mechanisms: reduction in cell damage from the sustained [Ca²⁺]_i and reduction of the inflammatory response. However, Orai1 has many roles outside the pancreas; thus inhibitors will likely have adverse effects limiting their therapeutic efficacy. Deletion of Orai1 in mice is embryonically lethal, and Orai1 has diverse roles in cardiovascular, neuronal, and muscular physiology, as well as platelet aggregation and the immune response. Loss-of-function mutations in ORAI1 cause severe immunodeficiency, tubular aggregate myopathy, and autoimmunity (4). Hence, any potential use of Orai1 blockers will require much caution. One potential solution would be a combination of TRPC channel blockers and low-dose Orai1 inhibitors to treat acute pancreatitis while minimizing the side effects due to inhibition of Orai1.

Small-molecule inhibitors have revolutionized the fields of oncology and immunology. Simultaneous blockade of multiple noxious stimuli is achieved by targeting intracellular signaling pathways, leading to improved disease outcomes. While the development of selective Ca²⁺ channel inhibitors is in its infancy, recent advances in the field have opened new possibilities that could similarly shift the treatment paradigm exocrine diseases.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

Author contributions: A.S., S.P., D.M.S., and S.M. edited and revised manuscript; A.S., S.P., D.M.S., and S.M. approved final version of manuscript; S.M. prepared figures; S.M. drafted manuscript.