Cyclic adenosine diphosphate ribose activates ryanodine receptors, whereas NAADP activates two-pore domain channels

The mechanism by which cyclic adenosine diphosphate ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP) mobilize intracellular Ca(2+) stores remains controversial. It is open to question whether cADPR regulates ryanodine receptors (RyRs) directly, as originally proposed, or indirectly by promoting Ca(2+) uptake into the sarco/endoplasmic reticulum by sarco/endoplasmic reticulum Ca(2+)-ATPases. Conversely, although we have proposed that NAADP mobilizes endolysosomal Ca(2+) stores by activating two-pore domain channels (TPCs), others suggest that NAADP directly activates RyRs. We therefore assessed Ca(2+) signals evoked by intracellular dialysis from a patch pipette of cADPR and NAADP into HEK293 cells that stably overexpress either TPC1, TPC2, RyR1, or RyR3. No change in intracellular Ca(2+) concentration was triggered by cADPR in either wild-type HEK293 cells (which are devoid of RyRs) or in cells that stably overexpress TPC1 and TPC2, respectively. By contrast, a marked Ca(2+) transient was triggered by cADPR in HEK293 cells that stably expressed RyR1 and RyR3. The Ca(2+) transient was abolished following depletion of endoplasmic reticulum stores by thapsigargin and block of RyRs by dantrolene but not following depletion of acidic Ca(2+) stores by bafilomycin. By contrast, NAADP failed to evoke a Ca(2+) transient in HEK293 cells that expressed RyR1 or RyR3, but it induced robust Ca(2+) transients in cells that stably overexpressed TPC1 or TPC2 and in a manner that was blocked following depletion of acidic stores by bafilomycin. We conclude that cADPR triggers Ca(2+) release by activating RyRs but not TPCs, whereas NAADP activates TPCs but not RyRs.     

Departure gate of acidic Ca2+ confirmed

More potent, but less known than IP₃ that liberates Ca²⁺ from the ER, NAADP releases Ca²⁺ from acidic stores. The notion that TPC channels mediate this Ca²⁺ release was questioned recently by studies suggesting that TPCs are rather PI(3,5)P₂‐activated Na⁺ channels. Ruas et al (2015) now partially reconcile these views by showing that TPCs significantly conduct both cations and confirm their activation by both NAADP and PI(3,5)P₂. They attribute the failure of others to observe TPC‐dependent NAADP‐induced Ca²⁺ release in vivo to inadequate mouse models that retain partial TPC function.     

Acidic NAADP-releasable Ca compartments in the megakaryoblastic cell line MEG01

Background

A novel family of intracellular Ca²⁺-release channels termed two-pore channels (TPCs) has been presented as the receptors of NAADP (nicotinic acid adenine dinucleotide phosphate), the most potent Ca²⁺ mobilizing intracellular messenger. TPCs have been shown to be exclusively localized to the endolysosomal system mediating NAADP-evoked Ca²⁺ release from the acidic compartments.

Objectives

The present study is aimed to investigate NAADP-mediated Ca²⁺ release from intracellular stores in the megakaryoblastic cell line MEG01.

Methods

Changes in cytosolic and intraluminal free Ca²⁺ concentrations were registered by fluorimetry using fura-2 and fura-ff, respectively; TPC expression was detected by PCR.

Results

Treatment of MEG01 cells with the H⁺/K⁺ ionophore nigericin or the V-type H⁺-ATPase selective inhibitor bafilomycin A1 revealed the presence of acidic Ca²⁺ stores in these cells, sensitive to the SERCA inhibitor 2,5-di-(tert-butyl)-1,4-hydroquinone (TBHQ). NAADP releases Ca²⁺ from acidic lysosomal-like Ca²⁺ stores in MEG01 cells probably mediated by the activation of TPC1 and TPC2 as demonstrated by TPC1 and TPC2 expression silencing and overexpression. Ca²⁺ efflux from the acidic lysosomal-like Ca²⁺ stores or the endoplasmic reticulum (ER) results in ryanodine-sensitive activation of Ca²⁺-induced Ca²⁺ release (CICR) from the complementary Ca²⁺ compartment.

Conclusion

Our results show for the first time NAADP-evoked Ca²⁺ release from acidic compartments through the activation of TPC1 and TPC2, and CICR, in a megakaryoblastic cell line.     

NAADP-induced intracellular calcium ion is mediated by the TPCs (two-pore channels) in hypoxia-induced pulmonary arterial hypertension

Pulmonary arterial hypertension (PAH) is a form of obstructive vascular disease. Chronic hypoxic exposure leads to excessive proliferation of pulmonary arterial smooth muscle cells and pulmonary arterial endothelial cells. This condition can potentially be aggravated by [Ca²⁺] _i mobilization. In the present study, hypoxia exposure of rat's model was established. Two-pore segment channels (TPCs) silencing was achieved in rats' models by injecting Lsh-TPC1 or Lsh-TPC2. The effects of TPC1/2 silencing on PAH were evaluated by H&E staining detecting pulmonary artery wall thickness and ELISA assay kit detecting NAADP concentrations in lung tissues. TPC1/2 silencing was achieved in PASMCs and PAECs, and cell proliferation was detected by MTT and BrdU incorporation assays. As the results shown, NAADP-activated [Ca²⁺]_i shows to be mediated via two-pore segment channels (TPCs) in PASMCs, with TPC1 being the dominant subtype. NAADP generation and TPC1/2 mRNA and protein levels were elevated in the hypoxia-induced rat PAH model; NAADP was positively correlated with TPC1 and TPC2 expression, respectively. In vivo, Lsh-TPC1 or Lsh-TPC2 infection significantly improved the mean pulmonary artery pressure and PAH morphology. In vitro, TPC1 silencing inhibited NAADP-AM-induced PASMC proliferation and [Ca²⁺]_i in PASMCs, whereas TPC2 silencing had minor effects during this process; TPC2 silencing attenuated NAADP-AM- induced [Ca²⁺]_i and ECM in endothelial cells, whereas TPC1 silencing barely ensued any physiological changes. In conclusion, TPC1/2 might provide a unifying mechanism within pulmonary arterial hypertension, which can potentially be regarded as a therapeutic target.     

TPC2 Is a Novel NAADP-sensitive Ca2+ Release Channel, Operating as a Dual Sensor of Luminal pH and Ca2+

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a molecule capable of initiating the release of intracellular Ca²⁺ required for many essential cellular processes. Recent evidence links two-pore channels (TPCs) with NAADP-induced release of Ca²⁺ from lysosome-like acidic organelles; however, there has been no direct demonstration that TPCs can act as NAADP-sensitive Ca²⁺ release channels. Controversial evidence also proposes ryanodine receptors as the primary target of NAADP. We show that TPC2, the major lysosomal targeted isoform, is a cation channel with selectivity for Ca²⁺ that will enable it to act as a Ca²⁺ release channel in the cellular environment. NAADP opens TPC2 channels in a concentration-dependent manner, binding to high affinity activation and low affinity inhibition sites. At the core of this process is the luminal environment of the channel. The sensitivity of TPC2 to NAADP is steeply dependent on the luminal [Ca²⁺] allowing extremely low levels of NAADP to open the channel. In parallel, luminal pH controls NAADP affinity for TPC2 by switching from reversible activation of TPC2 at low pH to irreversible activation at neutral pH. Further evidence earmarking TPCs as the likely pathway for NAADP-induced intracellular Ca²⁺ release is obtained from the use of Ned-19, the selective blocker of cellular NAADP-induced Ca²⁺ release. Ned-19 antagonizes NAADP-activation of TPC2 in a non-competitive manner at 1 μm but potentiates NAADP activation at nanomolar concentrations. This single-channel study provides a long awaited molecular basis for the peculiar mechanistic features of NAADP signaling and a framework for understanding how NAADP can mediate key physiological events.     

TPCs: Endolysosomal channels for Ca mobilization from acidic organelles triggered by NAADP

Two-pore channels (TPCs or TPCNs) are novel members of the large superfamily of voltage-gated cation channels with slightly higher sequence homology to the pore-forming subunits of voltage-gated Ca²⁺ and Na⁺ channels than most other members. Recent studies demonstrate that TPCs locate to endosomes and lysosomes and form Ca²⁺ release channels that respond to activation by the Ca²⁺ mobilizing messenger, nicotinic acid adenine dinucleotide phosphate (NAADP). With multiple endolysosomal targeted NAADP receptors now identified, important new insights into the regulation of endolysosomal function in health and disease will therefore be unveiled.     

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.     

Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) and Endolysosomal Two-pore Channels Modulate Membrane Excitability and Stimulus-Secretion Coupling in Mouse Pancreatic β Cells

Pancreatic β cells are electrically excitable and respond to elevated glucose concentrations with bursts of Ca²⁺ action potentials due to the activation of voltage-dependent Ca²⁺ channels (VDCCs), which leads to the exocytosis of insulin granules. We have examined the possible role of nicotinic acid adenine dinucleotide phosphate (NAADP)-mediated Ca²⁺ release from intracellular stores during stimulus-secretion coupling in primary mouse pancreatic β cells. NAADP-regulated Ca²⁺ release channels, likely two-pore channels (TPCs), have recently been shown to be a major mechanism for mobilizing Ca²⁺ from the endolysosomal system, resulting in localized Ca²⁺ signals. We show here that NAADP-mediated Ca²⁺ release from endolysosomal Ca²⁺ stores activates inward membrane currents and depolarizes the β cell to the threshold for VDCC activation and thereby contributes to glucose-evoked depolarization of the membrane potential during stimulus-response coupling. Selective pharmacological inhibition of NAADP-evoked Ca²⁺ release or genetic ablation of endolysosomal TPC1 or TPC2 channels attenuates glucose- and sulfonylurea-induced membrane currents, depolarization, cytoplasmic Ca²⁺ signals, and insulin secretion. Our findings implicate NAADP-evoked Ca²⁺ release from acidic Ca²⁺ storage organelles in stimulus-secretion coupling in β cells.     

NAADP-evoked Ca2+ signals through two-pore channel-1 require arginine residues in the first S4-S5 linker

Two-pore channels (TPCs) are two-domain members of the voltage-gated ion channel superfamily that localize to acidic organelles. Their mechanism of activation (ligands such as NAADP/PI(3,5)P₂ versus voltage) and ion selectivity (Ca²⁺ versus Na⁺) is debated. Here we report that a cluster of arginine residues in the first domain required for selective voltage-gating of TPC1 map not to the voltage-sensing fourth transmembrane region (S4) but to a cytosolic downstream region (S4-S5 linker). These residues are conserved between TPC isoforms suggesting a generic role in TPC activation. Accordingly, mutation of residues in TPC1 but not the analogous region in the second domain prevents Ca²⁺ release by NAADP in intact cells. Our data affirm the role of TPCs in NAADP-mediated Ca²⁺ signalling and unite differing models of channel activation through identification of common domain-specific residues.     

Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) and Ca2+ Mobilization

Many physiological processes are controlled by a great diversity of Ca^{2 +} signals that depend on Ca^{2 +} entry into the cell and/or Ca^{2 +} release from internal Ca^{2 +} stores. Ca^{2 +} mobilization from intracellular stores is gated by a family of messengers including inositol-1,4,5-trisphosphate (InsP₃), cyclic ADP-ribose (cADPR), and nicotinic acid adenine dinucleotide phosphate (NAADP). There is increasing evidence for a novel intracellular Ca^{2 +} release channel that may be targeted by NAADP and that displays properties distinctly different from the well-characterized InsP₃ and ryanodine receptors. These channels appear to localize on a wider range of intracellular organelles, including the acidic Ca^{2 +} stores. Activation of the NAADP-sensitive Ca^{2 +} channels evokes complex changes in cytoplasmic Ca^{2 +} levels by means of channel chatter with other intracellular Ca^{2 +} channels. The recent demonstration of changes in intracellular NAADP levels in response to physiologically relevant extracellular stimuli highlights the significance of NAADP as an important regulator of intracellular Ca^{2 +} signaling.     

Rapid Recycling of Ca2+ between IP3-Sensitive Stores and Lysosomes

Inositol 1,4,5-trisphosphate (IP₃) evokes release of Ca²⁺ from the endoplasmic reticulum (ER), but the resulting Ca²⁺ signals are shaped by interactions with additional intracellular organelles. Bafilomycin A₁, which prevents lysosomal Ca²⁺ uptake by inhibiting H⁺ pumping into lysosomes, increased the amplitude of the initial Ca²⁺ signals evoked by carbachol in human embryonic kidney (HEK) cells. Carbachol alone and carbachol in combination with parathyroid hormone (PTH) evoke Ca²⁺ release from distinct IP₃-sensitive Ca²⁺ stores in HEK cells stably expressing human type 1 PTH receptors. Bafilomycin A₁ similarly exaggerated the Ca²⁺ signals evoked by carbachol or carbachol with PTH, indicating that Ca²⁺ released from distinct IP₃-sensitive Ca²⁺ stores is sequestered by lysosomes. The Ca²⁺ signals resulting from store-operated Ca²⁺ entry, whether evoked by thapsigargin or carbachol, were unaffected by bafilomycin A₁. Using Gd³⁺ (1 mM) to inhibit both Ca²⁺ entry and Ca²⁺ extrusion, HEK cells were repetitively stimulated with carbachol to assess the effectiveness of Ca²⁺ recycling to the ER after IP₃-evoked Ca²⁺ release. Blocking lysosomal Ca²⁺ uptake with bafilomycin A₁ increased the amplitude of each carbachol-evoked Ca²⁺ signal without affecting the rate of Ca²⁺ recycling to the ER. This suggests that Ca²⁺ accumulated by lysosomes is rapidly returned to the ER. We conclude that lysosomes rapidly, reversibly and selectively accumulate the Ca²⁺ released by IP₃ receptors residing within distinct Ca²⁺ stores, but not the Ca²⁺ entering cells via receptor-regulated, store-operated Ca²⁺ entry pathways.     

Nicotinic Acid Adenine Dinucleotide Phosphate Plays a Critical Role in Naive and Effector Murine T Cells but Not Natural Regulatory T Cells

Nicotinic acid adenine dinucleotide phosphate (NAADP), the most potent Ca²⁺ mobilizing second messenger discovered to date, has been implicated in Ca²⁺ signaling in some lymphomas and T cell clones. In contrast, the role of NAADP in Ca²⁺ signaling or the identity of the Ca²⁺ stores targeted by NAADP in conventional naive T cells is less clear. In the current study, we demonstrate the importance of NAADP in the generation of Ca²⁺ signals in murine naive T cells. Combining live-cell imaging methods and a pharmacological approach using the NAADP antagonist Ned-19, we addressed the involvement of NAADP in the generation of Ca²⁺ signals evoked by TCR stimulation and the role of this signal in downstream physiological end points such as proliferation, cytokine production, and other responses to stimulation. We demonstrated that acidic compartments in addition to the endoplasmic reticulum were the Ca²⁺ stores that were sensitive to NAADP in naive T cells. NAADP was shown to evoke functionally relevant Ca²⁺ signals in both naive CD4 and naive CD8 T cells. Furthermore, we examined the role of this signal in the activation, proliferation, and secretion of effector cytokines by Th1, Th2, Th17, and CD8 effector T cells. Overall, NAADP exhibited a similar profile in mediating Ca²⁺ release in effector T cells as in their counterpart naive T cells and seemed to be equally important for the function of these different subsets of effector T cells. This profile was not observed for natural T regulatory cells.     

Expression of Ca2+-permeable two-pore channels rescues NAADP signalling in TPC-deficient cells

The second messenger NAADP triggers Ca²⁺ release from endo-lysosomes. Although two-pore channels (TPCs) have been proposed to be regulated by NAADP, recent studies have challenged this. By generating the first mouse line with demonstrable absence of both Tpcn1 and Tpcn2 expression (Tpcn1/2^−/−), we show that the loss of endogenous TPCs abolished NAADP-dependent Ca²⁺ responses as assessed by single-cell Ca²⁺ imaging or patch-clamp of single endo-lysosomes. In contrast, currents stimulated by PI(3,5)P₂ were only partially dependent on TPCs. In Tpcn1/2^−/− cells, NAADP sensitivity was restored by re-expressing wild-type TPCs, but not by mutant versions with impaired Ca²⁺-permeability, nor by TRPML1. Another mouse line formerly reported as TPC-null likely expresses truncated TPCs, but we now show that these truncated proteins still support NAADP-induced Ca²⁺ release. High-affinity [³²P]NAADP binding still occurs in Tpcn1/2^−/− tissue, suggesting that NAADP regulation is conferred by an accessory protein. Altogether, our data establish TPCs as Ca²⁺-permeable channels indispensable for NAADP signalling.     

Photoaffinity labeling of nicotinic acid adenine dinucleotide phosphate (NAADP) targets in mammalian cells

Nicotinic acid adenine dinucleotide phosphate (NAADP) is an agonist-generated second messenger that releases Ca(2+) from intracellular acidic Ca(2+) stores. Recent evidence has identified the two-pore channels (TPCs) within the endolysosomal system as NAADP-regulated Ca(2+) channels that release organellar Ca(2+) in response to NAADP. However, little is known about the mechanism coupling NAADP binding to calcium release. To identify the NAADP binding site, we employed a photoaffinity labeling method using a radioactive photoprobe based on 5-azido-NAADP ([(32)P-5N(3)]NAADP) that exhibits high affinity binding to NAADP receptors. In several systems that are widely used for studying NAADP-evoked Ca(2+) signaling, including sea urchin eggs, human cell lines (HEK293, SKBR3), and mouse pancreas, 5N(3)-NAADP selectively labeled low molecular weight sites that exhibited the diagnostic pharmacology of NAADP-sensitive Ca(2+) release. Surprisingly, we were unable to demonstrate labeling of endogenous, or overexpressed, TPCs. Furthermore, labeling of high affinity NAADP binding sites was preserved in pancreatic samples from TPC1 and TPC2 knock-out mice. These photolabeling data suggest that an accessory component within a larger TPC complex is responsible for binding NAADP that is unique from the core channel itself. This observation necessitates critical evaluation of current models of NAADP-triggered activation of the TPC family.     

TPC2 Proteins Mediate Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP)- and Agonist-evoked Contractions of Smooth Muscle

Agonists such as those acting at muscarinic receptors are thought to induce contraction of smooth muscle primarily through inositol 1,4,5-trisphosphate production and release of Ca²⁺ from sarcoplasmic reticulum. However, the additional Ca²⁺-mobilizing messengers cyclic adenosine diphosphate ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP) may also be involved in this process, the former acting on the sarcoplasmic reticulum, the latter acting on lysosome-related organelles. In this study, we provide the first systematic analysis of the capacity of inositol 1,4,5-trisphosphate, cADPR, and NAADP to cause contraction in smooth muscle. Using permeabilized guinea pig detrusor and taenia caecum, we show that all three Ca²⁺-mobilizing messengers cause contractions in both types of smooth muscle. We demonstrate that cADPR and NAADP play differential roles in mediating contraction in response to muscarinic receptor activation, with a sizeable role for NAADP and acidic calcium stores in detrusor muscle but not in taenia caecum, underscoring the heterogeneity of smooth muscle signal transduction systems. Two-pore channel proteins (TPCs) have recently been shown to be key components of the NAADP receptor. We show that contractile responses to NAADP were completely abolished, and agonist-evoked contractions were reduced and now became independent of acidic calcium stores in Tpcn2^−/− mouse detrusor smooth muscle. Our findings provide the first evidence that TPC proteins mediate a key NAADP-regulated tissue response brought about by agonist activation of a cell surface receptor.     

Acidic NAADP-sensitive Calcium Stores in the Endothelium: AGONIST-SPECIFIC RECRUITMENT AND ROLE IN REGULATING BLOOD PRESSURE*

Accumulating evidence implicates nicotinic acid adenine dinucleotide phosphate (NAADP) in the control of Ca²⁺-dependent functions. Little, however, is known concerning its role in the vascular endothelium, a major regulator of blood pressure. Here, we show that NAADP acetoxymethyl ester (NAADP-AM), a cell-permeant NAADP analog, increases cytosolic Ca²⁺ concentration in aortic endothelial cells. We demonstrate that these signals and those evoked by acetylcholine are blocked by disrupting acidic organelles with bafilomycin A1. In contrast, Ca²⁺ signals in response to thrombin are only partially inhibited by bafilomycin A1 treatment, and those to ATP were insensitive, suggesting that recruitment of acidic stores is agonist-specific. We further show that NAADP-evoked Ca²⁺ signals hyperpolarize endothelial cells and generate NO. Additionally, we demonstrate that NAADP dilates aortic rings in an endothelium- and NO-dependent manner. Finally, we show that intravenous administration of NAADP-AM into anesthetized rats decreases mean arterial pressure. Our data extend the actions of NAADP to the endothelium both in vitro and in vivo, pointing to a previously unrecognized role for this messenger in controlling blood pressure.     

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.     

Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP)-mediated Calcium Signaling and Arrhythmias in the Heart Evoked by β-Adrenergic Stimulation

Nicotinic acid adenine dinucleotide phosphate (NAADP) is the most potent Ca²⁺-releasing second messenger known to date. Here, we report a new role for NAADP in arrhythmogenic Ca²⁺ release in cardiac myocytes evoked by β-adrenergic stimulation. Infusion of NAADP into intact cardiac myocytes induced global Ca²⁺ signals sensitive to inhibitors of both acidic Ca²⁺ stores and ryanodine receptors and to NAADP antagonist BZ194. Furthermore, in electrically paced cardiac myocytes BZ194 blocked spontaneous diastolic Ca²⁺ transients caused by high concentrations of the β-adrenergic agonist isoproterenol. Ca²⁺ transients were recorded both as increases of the free cytosolic Ca²⁺ concentration and as decreases of the sarcoplasmic luminal Ca²⁺ concentration. Importantly, NAADP antagonist BZ194 largely ameliorated isoproterenol-induced arrhythmias in awake mice. We provide strong evidence that NAADP-mediated modulation of couplon activity plays a role for triggering spontaneous diastolic Ca²⁺ transients in isolated cardiac myocytes and arrhythmias in the intact animal. Thus, NAADP signaling appears an attractive novel target for antiarrhythmic therapy.     

NAADP influences excitation-contraction coupling by releasing calcium from lysosomes in atrial myocytes

In atrial myocytes, the sarcoplasmic reticulum (SR) has an essential role in regulating the force of contraction as a consequence of its involvement in excitation-contraction coupling (ECC). Nicotinic acid adenine dinucleotide phosphate (NAADP) is a Ca²⁺ mobilizing messenger that acts to release Ca²⁺ from an acidic store in mammalian cells. The photorelease of NAADP in atrial myocytes increased Ca²⁺ transient amplitude with no effect on accompanying action potentials or the L-type Ca²⁺ current. NAADP-AM, a cell permeant form of NAADP, increased Ca²⁺ spark amplitude and frequency. The effect on Ca²⁺ spark frequency could be prevented by bafilomycin A1, a vacuolar H⁺-ATPase inhibitor, or by disruption of lysosomes by GPN. Bafilomycin prevented staining of acidic stores with LysoTracker red by increasing lysosomal pH. NAADP-AM also produced an increase in the lysosomal pH, as detected by a reduction in LysoSensor green fluorescence. These effects of NAADP were associated with an increase in the amount of caffeine-releasable Ca²⁺ in the SR and may be regulated by β-adrenoceptor stimulation with isoprenaline. These observations are consistent with a role for NAADP in regulating ECC in atrial myocytes by releasing Ca²⁺ from an acidic store, which enhances SR Ca²⁺ release by increasing SR load.