NAADP and other Ca 2+ -mobilizing messengers are membrane impermeant and thus must be added directly to cell-free or broken-cell preparations to effect Ca 2+ release. The sea urchin egg homogenate, where the biological activity of NAADP was first reported, remains the gold standard cell-free system for studying NAADP-mediated Ca 2+ release. Here we describe how to prepare sea urchin egg homogenate and use it to measure NAADP-mediated Ca 2+ release.

In addition to mobilizing Ca²⁺, NAADP plays a role in modulating the luminal pH (pHL) of acidic stores of the endolysosomal system. The effects of NAADP on pHL have been most extensively studied in the sea urchin egg, both in the intact egg and in egg homogenates. Related observations have also been made in mammalian systems (e.g., guinea pig atrial myocytes and pancreatic acinar cells). Although the connection between Ca²⁺ mobilization and increase in pHL is not understood, pHL can be a useful parameter to measure when studying NAADP-mediated signaling. This protocol describes the fluorescent measurement of pHL of acidic stores. It relies on the use of acridine orange (AO), a standard dye for pHL. AO selectively accumulates to high concentrations in the lumen of organelles as a function of acidity; at these high concentrations it self-quenches. When pHL increases, some AO is lost from the vesicle. As a result, the lower luminal AO concentration relieves the quenching and fluorescence increases in the lumen.

Nicotinic acid adenine dinucleotide phosphate (NAADP), like the other major messengers for Ca²⁺ mobilization, is passively membrane-impermeant. Instead, a cell-permeant acetoxymethyl ester derivative of NAADP (NAADP-AM) can be synthesized as described here and used to study NAADP-mediated Ca²⁺ release.

Caged derivatives of Ca²⁺-mobilizing messengers, such as nicotinic acid adenine dinucleotide phosphate (NAADP), are particularly useful for establishing the effects of these messengers on Ca²⁺ signaling. Caged NAADP is no longer commercially available but can be synthesized in house, as described here. In brief, a stable precursor of the caging reagent is made and converted to an unstable reactive reagent immediately before addition to the compound to be caged.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a major messenger for Ca2+ mobilization in cells. NAADP-binding proteins are highly selective and have a strong affinity for NAADP. This is the basis of the radioreceptor binding assay, which is used to measure NAADP levels in cells and tissues and to identify cellular stimuli that use NAADP as an intracellular messenger. In the radioreceptor binding assay, radiolabeled NAADP ([32P]NAADP) competes with endogenous NAADP present in samples for binding to their receptors. Here, we describe the synthesis of [32P]NAADP for use in the radioreceptor binding assay.

Two-pore channels (TPCs) have been recently identified as NAADP-regulated Ca(2+) release channels, which are localized on the endolysosomal system. TPCs have a 12-transmembrane domain (TMD) structure and are evolutionary intermediates between the 24-TMD α-subunits of Na(+) or Ca(2+) channels and the transient receptor potential channel superfamily, which have six TMDs in a single subunit and form tetramers with 24 TMDs as active channels. Based on this relationship, it is predicted that TPCs dimerize to form functional channels, but the dimerization of human TPCs has so far not been studied. Using co-immunoprecipitation studies and a mass spectroscopic analysis of the immunocomplex, we show the presence of homo- and heteromeric complexes for human TPC1 and TPC2. Despite their largely distinct localization, we identified a discrete number of endosomes that coexpressed TPC1 and TPC2. Homo- and heteromerization were confirmed by a FRET study, showing that both proteins interacted in a rotational (N- to C-terminal/head-to-tail) symmetry. This is the first report describing the presence of homomultimeric TPC1 channels and the first study showing that TPCs are capable of forming heteromers.

Nicotinic acid dinucleotide phosphate (NAADP) is unique amongst Ca2+ mobilizing messengers in that its principal function is to mobilize Ca2+ from acidic organelles. Early studies indicated that it was likely that NAADP activates a novel Ca2+ release channel distinct from the well characterized Ca2+ release channels on the (sarco)-endoplasmic reticulum (ER), inositol trisphosphate and ryanodine receptors. In this review, we discuss the emergence of a novel family of endolysosomal channels, the two-pore channels (TPCs), as likely targets for NAADP, and how molecular and pharmacological manipulation of these channels is enhancing our understanding of the physiological roles of NAADP as an intracellular Ca2+ mobilizing messenger.

We have recently demonstrated that human TPC2 is an ion-channel equipped with the specialised gating and conduction properties required to fulfil the role of the lysosomal NAADP-sensitive Ca2+-release channel (Pitt et al., 2010 J.Biol.Chem. In press). We now examine the mechanisms that underlie NAADP activation of TPC2 channels after reconstitution of purified, recombinant TPC2 into artificial bilayers. We find that TPC2 channels gate in three basic modes: 1. The constitutively-active state, 2. The NAADP-activated state and 3. The NAADP-inactivated state. In the constitutively-active state, TPC2 can open, albeit with very low Po, in the apparent absence of activating ligands. Reducing cytosolic or luminal [Ca2+] from 10µM to <1nM does not abolish channel openings. The NAADP-activated state is observed at low [NAADP] (<1µM) and in this state, the affinity and efficacy of NAADP are crucially dependent on luminal pH and [Ca2+] whereas cytosolic Ca2+ (≤ 10 µM) does not increase Po. A unique characteristic of the NAADP-activated state is episodic gating where multiple channels open and close simultaneously in a synchronized manner. The NAADP-inactivated state is observed at high [NAADP], typically ≥1mM. In this state, constitutive channel openings are abolished and Po becomes zero indicating the presence of high affinity activation and lower affinity inactivation sites. An important finding of this study is that, unlike other major classes of Ca2+-release channel (RyR and IP3R), activation of TPC2 is not heavily dependent on a rise in cytosolic [Ca2+]. We predict that the unique, episodic coupled gating behaviour of TPC2 plays an important role in enabling TPC2 to provide a flexible Ca2+-release system capable of wide variations in the magnitude and time-dependence of Ca2+ fluxes from lysosomal stores. Supported by the BHF and Wellcome Trust.

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.

It has been suggested that two-pore domain channels (TPCs) are the NAADP receptors responsible for NAADP-mediated Ca2+-release from lysosome-related stores yet there is evidence that NAADP could also regulate RyR channels. We have therefore compared the effects of NAADP on native RyR1, RyR2 and purified human TPC2, reconstituted into artificial membranes under identical experimental conditions. Similar to RyR channels, we find that TPC2 behaves as an ion-channel permeable to both monovalent (300±14pS; symmetrical 210 mM K+; SD; n=3) and divalent cations (15±2pS; 10μM cis/50 mM trans Ca2+;SD; n=5) with no evidence for anion permeability (in a 210mM trans: 510mM cis KCl gradient, the reversal potential coincides with the calculated value for a channel ideally selective for cations (Erev=-23mV)). Addition of trans NAADP had no effect on TPC2, but cis application induced marked channel activation. In symmetrical 210mM K+ and 10μM Ca2+, NAADP dose-dependently activated TPC2 channels with an EC50 of 500nM. Addition of 200μM trans Ca2+ significantly increased the sensitivity of TPC2, shifting the EC50 to 5nM. We have previously demonstrated that ligand-activation of RyR channels is also highly sensitive to luminal Ca2+ and therefore we have investigated how NAADP affects RyR1 and RyR2 in the presence of sensitising levels of luminal Ca2+. Addition of NAADP (≤1μM) did not affect RyR2 Po but slightly activated RyR1 (1μM NAADP increased Po from 0.022±0.035 to 0.106±0.147; SD, n=5). In contrast, larger increases in TPC2 Po (0.001±0.002 to 0.4±0.2; SD, n=3, P<0.05) could be elicited with much lower NAADP concentrations (10nM). Our study is the first to show that animal TPCs form functional, Ca2+-permeable ion-channels. We also provide further evidence that TPC2 is capable of mediating NAADP-sensitive Ca2+-release from acidic organelles but do not rule out a role for RyR1. BHF supported