OCaR1 endows exocytic vesicles with autoregulatory competence by preventing uncontrolled Ca2+ release, exocytosis, and pancreatic tissue damage.

Regulated exocytosis is initiated by increased Ca2+ concentrations in close spatial proximity to secretory granules, which is effectively prevented when the cell is at rest. Here we showed that exocytosis of zymogen granules in acinar cells was driven by Ca2+ directly released from acidic Ca2+ stores including secretory granules through NAADP-activated two-pore channels (TPCs). We identified OCaR1 (encoded by Tmem63a) as an organellar Ca2+ regulator protein integral to the membrane of secretory granules that controlled Ca2+ release via inhibition of TPC1 and TPC2 currents. Deletion of OCaR1 led to extensive Ca2+ release from NAADP-responsive granules under basal conditions as well as upon stimulation of GPCR receptors. Moreover, OCaR1 deletion exacerbated the disease phenotype in murine models of severe and chronic pancreatitis. Our findings showed OCaR1 as a gatekeeper of Ca2+ release that endows NAADP-sensitive secretory granules with an autoregulatory mechanism preventing uncontrolled exocytosis and pancreatic tissue damage.

GABARAP sequesters the FLCN-FNIP tumor suppressor complex to couple autophagy with lysosomal biogenesis.

Adaptive changes in lysosomal capacity are driven by the transcription factors TFEB and TFE3 in response to increased autophagic flux and endolysosomal stress, yet the molecular details of their activation are unclear. LC3 and GABARAP members of the ATG8 protein family are required for selective autophagy and sensing perturbation within the endolysosomal system. Here, we show that during the conjugation of ATG8 to single membranes (CASM), Parkin-dependent mitophagy, and Salmonella-induced xenophagy, the membrane conjugation of GABARAP, but not LC3, is required for activation of TFEB/TFE3 to control lysosomal capacity. GABARAP directly binds to a previously unidentified LC3-interacting motif (LIR) in the FLCN/FNIP tumor suppressor complex and mediates sequestration to GABARAP-conjugated membrane compartments. This disrupts FLCN/FNIP GAP function toward RagC/D, resulting in impaired substrate-specific mTOR-dependent phosphorylation of TFEB. Thus, the GABARAP-FLCN/FNIP-TFEB axis serves as a molecular sensor that coordinates lysosomal homeostasis with perturbations and cargo flux within the autophagy-lysosomal network.

Estradiol analogs attenuate autophagy, cell migration and invasion by direct and selective inhibition of TRPML1, independent of estrogen receptors.

The cation channel TRPML1 is an important regulator of lysosomal function and autophagy. Loss of TRPML1 is associated with neurodegeneration and lysosomal storage disease, while temporary inhibition of this ion channel has been proposed to be beneficial in cancer therapy. Currently available TRPML1 channel inhibitors are not TRPML isoform selective and block at least two of the three human isoforms. We have now identified the first highly potent and isoform-selective TRPML1 antagonist, the steroid 17ß-estradiol methyl ether (EDME). Two analogs of EDME, PRU-10 and PRU-12, characterized by their reduced activity at the estrogen receptor, have been identified through systematic chemical modification of the lead structure. EDME and its analogs, besides being promising new small molecule tool compounds for the investigation of TRPML1, selectively affect key features of TRPML1 function: autophagy induction and transcription factor EB (TFEB) translocation. In addition, they act as inhibitors of triple-negative breast cancer cell migration and invasion.

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.

Mining of Ebola virus entry inhibitors identifies approved drugs as two-pore channel pore blockers.

Two-pore channels (TPCs) are Ca2+-permeable ion channels localised to the endo-lysosomal system where they regulate trafficking of various cargoes including viruses. As a result, TPCs are emerging as important drug targets. However, their pharmacology is ill-defined. There are no approved drugs to target them. And their mechanism of ligand activation is largely unknown. Here, we identify a number of FDA-approved drugs as TPC pore blockers. Using a model of the pore of human TPC2 based on recent structures of mammalian TPCs, we virtually screened a database of ~1500 approved drugs. Because TPCs have recently emerged as novel host factors for Ebola virus entry, we reasoned that Ebola virus entry inhibitors may exert their effects through inhibition of TPCs. Cross-referencing hits from the TPC virtual screen with two recent high throughput anti-Ebola screens yielded approved drugs targeting dopamine and estrogen receptors as common hits. These compounds inhibited endogenous NAADP-evoked Ca2+ release from sea urchin egg homogenates, NAADP-mediated channel activity of TPC2 re-routed to the plasma membrane, and PI(3,5)P2-mediated channel activity of TPC2 expressed in enlarged lysosomes. Mechanistically, single channel analyses showed that the drugs reduced mean open time consistent with a direct action on the pore. Functionally, drug potency in blocking TPC2 activity correlated with inhibition of Ebola virus-like particle entry. Our results expand TPC pharmacology through the identification of approved drugs as novel blockers, support a role for TPCs in Ebola virus entry, and provide insight into the mechanisms underlying channel regulation. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.

Opening the Orai1 gates.

How the store-operated channel Orai1 opens and the number of gates that control channel opening and conductance remain unclear. In this issue of Science Signaling, Frischauf et al report on the importance of the basic pore region in addition to the hydrophobic gate in channel gating and identify a site in transmembrane domain 2 (TM2) that controls pore opening.

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.

STIM-TRP Pathways and Microdomain Organization: Ca2+ Influx Channels: The Orai-STIM1-TRPC Complexes.

Ca2+ influx by plasma membrane Ca2+ channels is the crucial component of the receptor-evoked Ca2+ signal. The two main Ca2+ influx channels of non-excitable cells are the Orai and TRPC families of Ca2+ channels. These channels are activated in response to cell stimulation and Ca2+ release from the endoplasmic reticulum (ER). The protein that conveys the Ca2+ content of the ER to the plasma membrane is the ER Ca2+ sensor STIM1. STIM1 activates the Orai channels and is obligatory for channel opening. TRPC channels can function in two modes, as STIM1-dependent and STIM1-independent. When activated by STIM1, both channel types function at the ER/PM (plasma membrane) junctions. This chapter describes the properties and regulation of the channels by STIM1, with emphasis how and when TRPC channels function as STIM1-dependent and STIM1-independent modes and their unique Ca2+-dependent physiological functions that are not shared with the Orai channels.

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.

The CAR that drives Ca2+ to Orai1.

How Ca(2+) permeates the Orai1 channel and the mechanism by which the channel achieves high Ca(2+) selectivity remain critical questions in understanding store-operated Ca(2+) influx. In research published in Science Signaling, Frischauf et al. identified a Ca(2+)-accumulating region (CAR) in the extracellular opening of Orai1, which explains how concentrating Ca(2+) at the mouth of Orai1 facilitates channel permeation and contributes to selectivity.

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.

Essential roles for Cavß2 and Cav1 channels in thymocyte development and T cell homeostasis.

Calcium ions (Ca(2+)) are important in numerous signal transduction processes, including the development and differentiation of T cells in the thymus. We report that thymocytes have multiple types of pore-forming α subunits and regulatory ß subunits that constitute voltage-gated Ca(2+) (Cav) channels. In mice, T cell-specific deletion of the gene encoding the ß2 regulatory subunit of Cav channels (Cacnb2) reduced the abundances of the channels Cav1.2 and Cav1.3 (both of which contain pore-forming α1 subunits) and impaired T cell development, which led to a substantial decrease in the numbers of thymocytes and peripheral T cells. Similar to the effect of Cacnb2 deficiency, pharmacological blockade of pore-forming Cav1α subunits reduced the sustained Ca(2+) influx in thymocytes upon stimulation of the T cell receptor, decreased the abundance of the transcription factor NFATc3, inhibited the proliferation of thymocytes in vitro, and led to lymphopenia in mice. Together, our data suggest that Cav1 channels are conduits for the sustained Ca(2+) influx that is required for the development of T cells.

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.

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.

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. Here, we used homology modeling to identify a conserved STIM1(448-530) C-terminal inhibitory domain (CTID), whose deletion resulted in spontaneous clustering of STIM1 and full activation of Orai1 in the absence of store depletion. CTID regulated SCDI by determining access to and interaction of the STIM1 inhibitor SARAF with STIM1 Orai1 activation region (SOAR), the STIM1 domain that activates Orai1. CTID had two lobes, STIM1(448-490) and STIM1(490-530), with distinct roles in mediating access of SARAF to SOAR. The STIM1(448-490) lobe restricted, whereas the STIM1(490-530) lobe directed, SARAF to SOAR. The two lobes cooperated to determine the features of SCDI. These findings highlight the central role of STIM1 in SCDI and provide a molecular mechanism for SCDI of Orai1.

The energetic consequences of loop 9 gating motions in acetylcholine receptor-channels.

Acetylcholine receptor-channels (AChRs) mediate fast synaptic transmission between nerve and muscle. In order to better-understand the mechanism by which this protein assembles and isomerizes between closed- and open-channel conformations we measured changes in the diliganded gating equilibrium constant (E(2)) consequent to mutations of residues at the C-terminus of loop 9 (L9) in the α and ε subunits of mouse neuromuscular AChRs. These amino acids are close to two interesting interfaces, between the extracellular and transmembrane domain within a subunit (E­T interface) and between primary and complementary subunits (P­C interface). Most α subunit mutations modestly decreased E(2) (mainly by slowing the channel-opening rate constant) and sometimes produced AChRs that had heterogeneous gating kinetic properties. Mutations in the ε subunit had a larger effect and could either increase or decrease E(2), but did not induce kinetic heterogeneity. There are broad-but-weak energetic interactions between αL9 residues and others at the αE­T interface, as well as between the εL9 residue and others at the P­C interface (in particular, the M2­M3 linker). These interactions serve, in part, to maintain the structural integrity of the AChR assembly at the E­T interface. Overall, the energy changes of L9 residues are significant but smaller than in other regions of the protein.

Intraspecific aflatoxin inhibition in Aspergillus flavus is thigmoregulated, independent of vegetative compatibility group and is strain dependent.

Biological control of preharvest aflatoxin contamination by atoxigenic stains of Aspergillus flavus has been demonstrated in several crops. The assumption is that some form of competition suppresses the fungus's ability to infect or produce aflatoxin when challenged. Intraspecific aflatoxin inhibition was demonstrated by others. This work investigates the mechanistic basis of that phenomenon. A toxigenic and atoxigenic isolate of A. flavus which exhibited intraspecific aflatoxin inhibition when grown together in suspended disc culture were not inhibited when grown in a filter insert-plate well system separated by a .4 or 3 µm membrane. Toxigenic and atoxigenic conidial mixtures (50∶50) placed on both sides of these filters restored inhibition. There was ∼50% inhibition when a 12 µm pore size filter was used. Conidial and mycelial diameters were in the 3.5-7.0 µm range and could pass through the 12 µm filter. Larger pore sizes in the initially separated system restored aflatoxin inhibition. This suggests isolates must come into physical contact with one another. This negates a role for nutrient competition or for soluble diffusible signals or antibiotics in aflatoxin inhibition. The toxigenic isolate was maximally sensitive to inhibition during the first 24 hrs of growth while the atoxigenic isolate was always inhibition competent. The atoxigenic isolate when grown with a green fluorescent protein (GFP) toxigenic isolate failed to inhibit aflatoxin indicating that there is specificity in the touch inhibiton. Several atoxigenic isolates were found which inhibited the GFP isolate. These results suggest that an unknown signaling pathway is initiated in the toxigenic isolate by physical interaction with an appropriate atoxigenic isolate in the first 24 hrs which prevents or down-regulates normal expression of aflatoxin after 3-5 days growth. We suspect thigmo-downregulation of aflatoxin synthesis is the mechanistic basis of intraspecific aflatoxin inhibition and the major contributor to biological control of aflatoxin contamination.

Transient receptor potential mucolipin 1 (TRPML1) and two-pore channels are functionally independent organellar ion channels.

NAADP is a potent second messenger that mobilizes Ca(2+) from acidic organelles such as endosomes and lysosomes. The molecular basis for Ca(2+) release by NAADP, however, is uncertain. TRP mucolipins (TRPMLs) and two-pore channels (TPCs) are Ca(2+)-permeable ion channels present within the endolysosomal system. Both have been proposed as targets for NAADP. In the present study, we probed possible physical and functional association of these ion channels. Exogenously expressed TRPML1 showed near complete colocalization with TPC2 and partial colocalization with TPC1. TRPML3 overlap with TPC2 was more modest. TRPML1 and to some extent TRPML3 co-immunoprecipitated with TPC2 but less so with TPC1. Current recording, however, showed that TPC1 and TPC2 did not affect the activity of wild-type TRPML1 or constitutively active TRPML1(V432P). N-terminally truncated TPC2 (TPC2delN), which is targeted to the plasma membrane, also failed to affect TRPML1 and TRPML1(V432P) channel function or TRPML1(V432P)-mediated Ca(2+) influx. Whereas overexpression of TPCs enhanced NAADP-mediated Ca(2+) signals, overexpression of TRPML1 did not, and the dominant negative TRPML1(D471K) was without affect on endogenous NAADP-mediated Ca(2+) signals. Furthermore, the single channel properties of NAADP-activated TPC2delN were not affected by TRPML1. Finally, NAADP-evoked Ca(2+) oscillations in pancreatic acinar cells were identical in wild-type and TRPML1(-/-) cells. We conclude that although TRPML1 and TPCs are present in the same complex, they function as two independent organellar ion channels and that TPCs, not TRPMLs, are the targets for NAADP.