Archana Jha

Convergent regulation of the lysosomal two‐pore channel‐2 by Mg2+, NAADP, PI(3,5)P2 and multiple protein kinases

Lysosomal Ca2+ homeostasis is implicated in disease and controls many lysosomal functions. A key in understanding lysosomal Ca2+ 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 Mg2+ and the MAPKs, JNK and P38 as novel regulators of TPC2. Cytoplasmic Mg2+ specifically inhibited TPC2 outward current, whereas lysosomal Mg2+ partially inhibited both outward and inward currents in a lysosomal lumen pH‐dependent manner. Under controlled Mg2+, 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 Ca2+ release in intact cells is regulated by Mg2+, 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 Ca2+ signaling and link this pathway to Mg2+ homeostasis and MAP kinases, pointing to roles for lysosomal Ca2+ in cell growth, inflammation and cancer.

Transient Receptor Potential Mucolipin 1 (TRPML1) and Two-pore Channels Are Functionally Independent Organellar Ion Channels

NAADP is a potent second messenger that mobilizes Ca2+ from acidic organelles such as endosomes and lysosomes. The molecular basis for Ca2+ release by NAADP, however, is uncertain. TRP mucolipins (TRPMLs) and two-pore channels (TPCs) are Ca2+-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 Ca2+ influx. Whereas overexpression of TPCs enhanced NAADP-mediated Ca2+ signals, overexpression of TRPML1 did not, and the dominant negative TRPML1(D471K) was without affect on endogenous NAADP-mediated Ca2+ signals. Furthermore, the single channel properties of NAADP-activated TPC2delN were not affected by TRPML1. Finally, NAADP-evoked Ca2+ 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.

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

Two distinct lobes in the C-terminal inhibitory domain in STIM1 determine access of the inhibitor SARAF to the activating SOAR domain to regulate the slow Ca2+-dependent inactivation of Orai1.

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. 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 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.

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.

How does NAADP release lysosomal Ca2

paper type 174 Channels Volume 8 Issue 3 CoMMentary Ca 2+ is a ubiquitous yet unusual second messenger in that it is not metabo-lized. Instead, Ca 2+ levels in the cytosol are regulated by dynamic redistribution of Ca 2+ across the plasma membrane and the membranes of organelles. In addition to the ER, acidic organelles such as lyso-somes, endosomes, and secretory vesicles (in secretory cell types) store and release Ca 2+. Endolysosomal Ca 2+ homeosta-sis has several cellular functions; among them are apoptosis, trafficking, energy metabolism and fusion/fission events. The messenger NAADP appears particularly important in mobilizing acidic Ca 2+ stores and in many cases NAADP-evoked signals are amplified by Ca 2+ channels on the ER.

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.

STIM-TRP Pathways and Microdomain Organization: Ca 2+ 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.

Lipids at membrane contact sites: cell signaling and ion transport

Communication between organelles is essential to coordinate cellular functions and the cells 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.