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Melanosomal pH is important for the synthesis of melanin as the rate-limiting enzyme, tyrosinase, is very pH-sensitive. The soluble adenylyl cyclase (sAC) signaling pathway was recently identified as a regulator of melanosomal pH in melanocytes; however, the melanosomal proteins critical for sAC-dependent regulation of melanosomal pH were undefined. We now systematically examine four well-characterized melanosomal membrane proteins to determine whether any of them are required for sAC-dependent regulation of melanosomal pH. We find that OA1, OCA2, and SLC45A2 are dispensable for sAC-dependent regulation of melanosomal pH. In contrast, TPC2 activity is required for sAC-dependent regulation of melanosomal pH and melanin synthesis. In addition, activation of TPC2 by NAADP-AM rescues melanosomal pH alkalinization and reduces melanin synthesis following pharmacologic or genetic inhibition of sAC signaling. These studies establish TPC2 as a critical melanosomal protein for sAC-dependent regulation of melanosomal pH and pigmentation.

Two-pore channels and TRP mucolipins are ubiquitous endo-lysosomal cation channels of pathophysiological relevance. Both are Ca2+-permeable and regulated by phosphoinositides, principally PI(3,5)P2. Accumulating evidence has uncovered synergistic channel activation by PI(3,5)P2 and endogenous metabolites such as the Ca2+ mobilizing messenger NAADP, synthetic agonists including approved drugs and physical cues such as voltage and osmotic pressure. Here, we provide an overview of this coordination.

UNASSIGNED: CD38 is a multifunctional enzyme with a potent Ca2+ mobilizing effect, cyclic ADP-ribose (cADPR), and nicotinic acid adenine dinucleotide phosphate (NAADP). Here, we aimed to demonstrate the role of CD38 in platelets via protein kinase C (PKC)-mediated internalization and activation.
UNASSIGNED: Mouse platelets were used in this study. Thrombin, an agonist of platelet function, provoked a prompt and long-lasting increase in intracellular Ca2+ concentration ([Ca2+]i), resulting from an interplay of multifold Ca2+ mobilizing messengers.The signaling pathway was delineated using different inhibitors and techniques such as platelet aggregation assay, intracellular calcium measurements, immunoprecipitation, immunoblotting, and flow cytometry.
UNASSIGNED: We observed a sequential formation of cADPR and NAADP through CD38 activation by PKC of non-muscle myosin heavy chain IIA (MHCIIA), resulting in phospholipase C (PLC) activation in the thrombin-stimulated platelets. These findings reveal that PKC is fundamental in activating CD38 and elicits a physiological response in the murine platelets.
UNASSIGNED: PKC is involved in many signaling pathways. Specifically, PKC is involved in the internalization of CD38 via MHCIIA in CD38+/+ wild-type (WT) and CD38-/- knockout mice (KO). CD38 generates calcium-mobilizing agents that act on specific receptors of the calcium stores. Calcium triggered platelet aggregation while serving as a secondary messenger.

The recent elegant study by Y. Yuan and colleagues examined functional relationships between the lysosomal two-pore channels 2 (TPC2) and IP3 receptors (IP3Rs) located in the endoplasmic reticulum [1]. The findings of this study suggest functional coupling of these channels and receptors. The study also describes interesting novel phenomena, which may indicate an additional coupling mechanism.

Nicotinic acid adenine dinucleotide phosphate (NAADP), identified as one of the most potent calcium-mobilizing second messengers, has been studied in different eukaryotic cell types, including lymphocytes. Although aspects of NAADP-mediated calcium release in lymphocytes are still under debate, the organelles pertaining to NAADP-mediated calcium release are often characterized as acidic and related to lysosomes. Although NAADP-mediated calcium release in different subsets of T cells, including naïve, effector and natural regulatory T cells, has been studied, it has not been widely studied in memory CD4+ T cells, which show a different calcium flux profile. Using a pharmacological approach, the effect of Ned-19, an NAADP pathway antagonist, on the involvement of NAADP in TCR activation in murine memory CD4+ T cells and their downstream effector functions, such as proliferation and cytokine production, was studied. According to this study, Ned-19 inhibited TCR-mediated calcium flux and its downstream effector functions in primary memory CD4+ T cells. The study also revealed that both extracellular and intracellular calcium stores, including endoplasmic reticulum and lysosome-like acidic calcium stores, contribute to the TCR-mediated calcium flux in memory CD4+ T cells. NAADP-AM, a cell permeable analogue of NAADP, was shown to release calcium in memory CD4+ T cells and calcium flux was inhibited by Ned-19.

Bafilomycin A1 inhibits V-type H+ ATPases on the molecular level, which acidifies endo-lysosomes. The main objective of the study was to assess the effect of bafilomycin A1 on Ca2+ content, NAADP-induced Ca2+ release, and ATPase activity in rat hepatocytes and human colon cancer samples. Chlortetracycline (CTC) was used for a quantitative measure of stored calcium in permeabilized rat hepatocytes. ATPase activity was determined by orthophosphate content released after ATP hydrolysis in subcellular post-mitochondrial fraction obtained from rat liver as well as from patients\' samples of colon mucosa and colorectal cancer samples. In rat hepatocytes, bafilomycin A1 decreased stored Ca2+ and prevented the effect of NAADP on stored Ca2+. This effect was dependent on EGTA-Ca2+ buffers in the medium. Bafilomycin A1 significantly increased the activity of Ca2+ ATPases of endoplasmic reticulum (EPR), but not plasma membrane (PM) Ca2+ ATPases in rat liver. Bafilomycin A1 also prevented the effect of NAADP on these pumps. In addition, bafilomycin A1 reduced Na+/K+ ATPase activity and increased basal Mg2+ ATPase activity in the subcellular fraction of rat liver. Concomitant administration of bafilomycin A1 and NAADP enhanced these effects. Bafilomycin A1 increased the activity of the Ca2+ ATPase of EPR in the subcellular fraction of normal human colon mucosa and also in colon cancer tissue samples. In contrast, it decreased Ca2+ ATPase PM activity in samples of normal human colon mucosa and caused no changes in colon cancer. Bafilomycin A1 decreased Na+/K+ ATPase activity and increased basal Mg2+ ATPase activity in normal colon mucosa samples and in human colon cancer samples. It can be concluded that bafilomycin A1 targets NAADP-sensitive acidic Ca2+ stores, effectively modulates ATPase activity, and assumes the link between acidic stores and EPR. Bafilomycin A1 may be useful for cancer therapy.

Lysosomes and the endoplasmic reticulum (ER) are Ca2+ stores mobilized by the second messengers NAADP and IP3, respectively. Here, we establish Ca2+ signals between the two sources as fundamental building blocks that couple local release to global changes in Ca2+. Cell-wide Ca2+ signals evoked by activation of endogenous NAADP-sensitive channels on lysosomes comprise both local and global components and exhibit a major dependence on ER Ca2+ despite their lysosomal origin. Knockout of ER IP3 receptor channels delays these signals, whereas expression of lysosomal TPC2 channels accelerates them. High-resolution Ca2+ imaging reveals elementary events upon TPC2 opening and signals coupled to IP3 receptors. Biasing TPC2 activation to a Ca2+-permeable state sensitizes local Ca2+ signals to IP3. This increases the potency of a physiological agonist to evoke global Ca2+ signals and activate a downstream target. Our data provide a conceptual framework to understand how Ca2+ release from physically separated stores is coordinated.

Ca2+ signaling is essential for cardiac contractility and excitability in heart function and remodeling. Intriguingly, little is known about the role of a new family of ion channels, the endo-lysosomal non-selective cation \"two-pore channel\" (TPCs) in heart function. Here we have used double TPC knock-out mice for the 1 and 2 isoforms of TPCs (Tpcn1/2-/-) and evaluated their cardiac function. Doppler-echocardiography unveils altered left ventricular (LV) systolic function associated with a LV relaxation impairment. In cardiomyocytes isolated from Tpcn1/2-/- mice, we observed a reduction in the contractile function with a decrease in the sarcoplasmic reticulum Ca2+ content and a reduced expression of various key proteins regulating Ca2+ stores, such as calsequestrin. We also found that two main regulators of the energy metabolism, AMP-activated protein kinase and mTOR, were down regulated. We found an increase in the expression of TPC1 and TPC2 in a model of transverse aortic constriction (TAC) mice and in chronically isoproterenol infused WT mice. In this last model, adaptive cardiac hypertrophy was reduced by Tpcn1/2 deletion. Here, we propose a central role for TPCs and lysosomes that could act as a hub integrating information from the excitation-contraction coupling mechanisms, cellular energy metabolism and hypertrophy signaling.

Membrane contact sites (MCSs) between endosomes and the endoplasmic reticulum (ER) are thought to act as specialized trigger zones for Ca2+ signaling, where local Ca2+ released via endolysosomal ion channels is amplified by ER Ca2+-sensitive Ca2+ channels into global Ca2+ signals. Such amplification is integral to the action of the second messenger, nicotinic acid adenine dinucleotide phosphate (NAADP). However, functional regulators of inter-organellar Ca2+ crosstalk between endosomes and the ER remain poorly defined. Here, we identify progesterone receptor membrane component 1 (PGRMC1), an ER transmembrane protein that undergoes a unique heme-dependent dimerization, as an interactor of the endosomal two pore channel, TPC1. NAADP-dependent Ca2+ signals were potentiated by PGRMC1 overexpression through enhanced functional coupling between endosomal and ER Ca2+ stores and inhibited upon PGRMC1 knockdown. Point mutants in PGMRC1 or pharmacological manipulations that reduced its interaction with TPC1 were without effect. PGRMC1 therefore serves as a TPC1 interactor that regulates ER-endosomal coupling with functional implications for cellular Ca2+ dynamics and potentially the distribution of heme.