The synthesis and characterization of a clickable-photoactive NAADP analog active in human cells

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent Ca²⁺ mobilizing second messenger which triggers Ca²⁺ release in both sea urchin egg homogenates and in mammalian cells. The NAADP binding protein has not been identified and the regulation of NAADP mediated Ca²⁺ release remains controversial. To address this issue, we have synthesized an NAADP analog in which 3-azido-5-azidomethylbenzoic acid is attached to the amino group of 5-(3-aminopropyl)-NAADP to produce an NAADP analog which is both a photoaffinity label and clickable. This ‘all-in-one-clickable’ NAADP (AIOC-NAADP) elicited Ca²⁺ release when microinjected into cultured human SKBR3 cells at low concentrations. In contrast, it displayed little activity in sea urchin egg homogenates where very high concentrations were required to elicit Ca²⁺ release. In mammalian cell homogenates, incubation with low concentrations of [³²P]AIOC-NAADP followed by irradiation with UV light resulted in labeling 23 kDa protein(s). Competition between [³²P]AIOC-NAADP and increasing concentrations of NAADP demonstrated that the labeling was selective. We show that this label recognizes and selectively photodervatizes the 23 kDa NAADP binding protein(s) in cultured human cells identified in previous studies using [³²P]5-N₃-NAADP.     

Fluorescent analogs of NAADP with calcium mobilizing activity

Nicotinic acid adenine dinucleotide phosphate (NAADP) mobilizes Ca²⁺ through a mechanism totally independent of cyclic ADP-ribose or inositol trisphosphate. Fluorescent analogs of NAADP were synthesized in this study to facilitate further characterization of this novel Ca²⁺ release mechanism. The base-exchange reaction catalyzed by ADP-ribosyl cyclase was utilized to convert nicotinamide 1,N⁶-ethenoadenine dinucleotide phosphate to a fluorescent product, nicotinic acid 1,N⁶-ethenoadenine dinucleotide phosphate (etheno-NAADP). The excitation spectrum of the product showed two maxima at 275 nm and 300 nm and an emission maximum at 410 nm. An aza derivative of etheno-NAADP was also synthesized by sequential treatments with NaOH and nitrite. The product, nicotinic acid 1,N⁶-etheno-2-aza-adenine dinucleotide phosphate (etheno-aza-NAADP) had excitation maxima at 280 nm and 360 nm and an emission maximum at 470 nm. The fluorescence of both analogs was sensitive to polarity and exhibited a 3–4-fold enhancement going from an aqueous buffer to an organic solvent. Proton-NMR measurements confirmed the presence of the etheno ring in both analogs. In the aza derivative the proton at the 2-position of the adenine ring was absent, consistent with the conversion of the 2-carbon to a nitrogen. Both analogs could activate Ca²⁺ release from sea urchin egg homogenates and the half-maximal concentrations for etheno-aza-NAADP and etheno-NAADP were at about 2.5 μM and 5 μM, respectively. At sub-threshold concentrations, both analogs could also function as antagonists, inactivating the NAADP-sensitive Ca²⁺ release with a half-maximal concentration of 60–80 nM. Microinjection of etheno-aza-NAADP into live eggs activated Ca²⁺ increase and triggered a cortical exocytotic reaction confirming its effectiveness in vivo. These fluorescent analogs are potentially useful for visualizing the novel Ca²⁺ stores that are sensitive to NAADP in live cells.     

5-Azido-8-ethynyl-NAADP: A bifunctional,clickable photoaffinity probe for the identification of NAADP receptors

Nicotinic acid adenine dinucleotide phosphate is an evolutionarily conserved second messenger, which mobilizes Ca²⁺ from acidic stores. The molecular identity of the NAADP receptor has yet to be defined. In pursuit of isolating and identifying NAADP-binding proteins, we synthesized and characterized a bifunctional probe that incorporates both a photoactivatable crosslinking azido moiety at the 5-position of the nicotinic ring and a ‘clickable’ ethynyl moiety to the 8-adenosyl position in NAADP. Microinjection of this 5N₃-8-ethynyl-NAADP into cultured U2OS cells induced robust Ca²⁺ responses. Higher concentrations of 5N₃-8-ethynyl were required to elicit Ca²⁺ release or displace ³²P-NAADP in radioligand binding experiments in sea urchin egg homogenates. In human cell extracts, incubation of ³²P-5N₃-8-ethynyl-NAADP followed by UV irradiation resulted in selective labeling of 23 kDa and 35 kDa proteins and photolabeling of these proteins was prevented when incubated in the presence of unlabeled NAADP. Compared to the monofunctional ³²P-5N₃-NAADP, the clickable ³²P-5N₃-8-ethynyl-NAADP demonstrated less labeling of the 23 kDa and 35 kDa proteins (~3-fold) but provided an opportunity for further enrichment through the ‘clickable’ ethynyl moiety. No proteins were specifically labeled by ³²P-5N₃-8-ethynyl-NAADP in sea urchin egg homogenate. These experiments demonstrate that 5N₃-8-ethynyl-NAADP is biologically active and selectively labels putative NAADP-binding proteins in mammalian systems, evidencing a ‘bifunctional’ probe with utility for isolating NAADP-binding proteins.     

Photoaffinity labeling of high affinity nicotinic acid adenine dinucleotide phosphate (NAADP)-binding proteins in sea urchin egg

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a messenger that regulates calcium release from intracellular acidic stores. Recent studies have identified two-pore channels (TPCs) as endolysosomal channels that are regulated by NAADP; however, the nature of the NAADP receptor binding site is unknown. To further study NAADP binding sites, we have synthesized and characterized [(32)P-5-azido]nicotinic acid adenine dinucleotide phosphate ([(32)P-5N(3)]NAADP) as a photoaffinity probe. Photolysis of sea urchin egg homogenates preincubated with [(32)P-5N(3)]NAADP resulted in specific labeling of 45-, 40-, and 30-kDa proteins, which was prevented by inclusion of nanomolar concentrations of unlabeled NAADP or 5N(3)-NAADP, but not by micromolar concentrations of structurally related nucleotides such as NAD, nicotinic acid adenine dinucleotide, nicotinamide mononucleotide, nicotinic acid, or nicotinamide. [(32)P-5N(3)]NAADP binding was saturable and displayed high affinity (K(d) ～10 nM) in both binding and photolabeling experiments. [(32)P-5N(3)]NAADP photolabeling was irreversible in a high K(+) buffer, a hallmark feature of NAADP binding in the egg system. The proteins photolabeled by [(32)P-5N(3)]NAADP have molecular masses smaller than the sea urchin TPCs, and antibodies to TPCs do not detect any immunoreactivity that comigrates with either the 45-kDa or the 40-kDa photolabeled proteins. Interestingly, antibodies to TPC1 and TPC3 were able to immunoprecipitate a small fraction of the 45- and 40-kDa photolabeled proteins, suggesting that these proteins associate with TPCs. These data suggest that high affinity NAADP binding sites are distinct from TPCs.     

NAADP receptors

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a recently described Ca2+ mobilizing messenger. First described in the sea urchin egg, it has been shown to mobilize Ca2+ from intracellular stores. It is a remarkably potent molecule, and recent reports show that its cellular levels change in response to a variety of agonists confirming its role as a Ca2+ mobilizing messenger. In many cases NAADP interacts with other Ca2+ mobilizing messengers such as inositol 1,4,5 trisphosphate (IP3 and cyclic adenosine diphosphate ribose (cADPR) in shaping cytosolic Ca2+ signals. What is not clear is the molecular nature of the NAADP-sensitive Ca2+ release mechanism and its sub-cellular localization. In this review we focus on the recent progress made in sea urchin eggs, which indicates that NAADP activates a novel Ca2+ release channel distinct from the relatively well-characterized IP3 and ryanodine receptors. Furthermore, in the sea urchin egg, the NAADP-sensitive store appears to be separate from the endoplasmic reticulum (ER) and is most likely an acidic store. These findings have also been reinforced by similar findings by some in mammalian cells. Finally, we discuss ongoing strategies to characterise NAADP-binding proteins which will greatly enhance our understanding of NAADP-mediated Ca2+ signalling, and lead to the development of more selective tools to probe the role of this messenger.     

Role of the nicotinic acid group in NAADP receptor selectivity

Nicotinic acid adenine dinucleotide phosphate (NAADP) has been shown to be an intracellular Ca2+-releasing messenger in a wide variety of systems to date. Its actions are both potent and highly specific despite differing structurally from the endogenous cellular co-factor and its precursor, NADP, only in the substitution of a hydroxyl for the amine group at the 3' position of the pyridine ring. This substitution allows NAADP to bind to a membrane-localized binding site in sea urchin egg homogenates with an IC50 at least 1000-fold greater than that of NADP as measured by competition radioligand binding assays. This suggests that the NAADP receptor protein must include certain features in the NAADP binding site that regulate this specificity. In order to investigate this interaction, we synthesised a series of NAADP analogues differing from NAADP at the 3' position of the pyridine ring that included both simple carboxylic acid analogues as well as a series of chemical isosters. We then investigated both their affinity for the NAADP binding site in sea urchin egg homogenates and their ability to activate the NAADP sensitive Ca2+ channel. We hereby show that a negative charge at the 3' position is an important determinant of affinity but the protein displays a large tolerance for the size of the group. Furthermore, the protein does not easily accommodate multiple charged groups or large uncharged groups.     

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.     

Solubilization of receptors for the novel Ca2+-mobilizing messenger,nicotinic acid adenine dinucleotide phosphate

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent Ca(2+) mobilizing agent in a variety of broken and intact cell preparations. In sea urchin egg homogenates, NAADP releases Ca(2+) independently of inositol trisphosphate or ryanodine receptor activation. Little, however, is known concerning the molecular target for NAADP. Here we report for the first time solubilization of NAADP receptors from sea urchin egg homogenates. Supernatant fractions, prepared following Triton X-100 treatment, bound [(32)P]NAADP with similar affinity and selectivity as membrane preparations. Furthermore, the unusual non-dissociating nature of NAADP binding to its receptor was preserved upon solubilization. NAADP receptors could also be released into supernatant fractions upon detergent treatment of membranes prelabeled with [(32)P]NAADP. Tagged receptors prepared in this way, were readily resolved by native gel electrophoresis as a single protein target. Gel filtration and sucrose density gradient centrifugation analysis indicates that NAADP receptors are substantially smaller than inositol trisphosphate or ryanodine receptors, providing further biochemical evidence that NAADP activates a novel intracellular Ca(2+) release channel.     

Analogues of the Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) Antagonist Ned-19 Indicate Two Binding Sites on the NAADP Receptor

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a Ca²⁺-releasing messenger. Biological data suggest that its receptor has two binding sites: one high-affinity locking site and one low-affinity opening site. To directly address the presence and function of these putative binding sites, we synthesized and tested analogues of the NAADP antagonist Ned-19. Ned-19 itself inhibits both NAADP-mediated Ca²⁺ release and NAADP binding. A fluorometry bioassay was used to assess NAADP-mediated Ca²⁺ release, whereas a radioreceptor assay was used to assess binding to the NAADP receptor (only at the high-affinity site). In Ned-20, the fluorine is para rather than ortho as in Ned-19. Ned-20 does not inhibit NAADP-mediated Ca²⁺ release but inhibits NAADP binding. Conversely, Ned-19.4 (a methyl ester of Ned-19) inhibits NAADP-mediated Ca²⁺ release but cannot inhibit NAADP binding. Furthermore, Ned-20 prevents the self-desensitization response characteristic of NAADP in sea urchin eggs, confirming that this response is mediated by a high-affinity allosteric site to which NAADP binds in the radioreceptor assay. Collectively, these data provide the first direct evidence for two binding sites (one high- and one low-affinity) on the NAADP receptor.     

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.     

A screening campaign in sea urchin egg homogenate as a platform for discovering modulators of NAADP-dependent Ca2+ signaling in human cells

The Ca²⁺ mobilizing second messenger nicotinic acid adenine dinucleotide phosphate (NAADP) regulates intracellular trafficking events, including translocation of certain enveloped viruses through the endolysosomal system. Targeting NAADP-evoked Ca²⁺ signaling may therefore be an effective strategy for discovering novel antivirals as well as therapeutics for other disorders. To aid discovery of novel scaffolds that modulate NAADP-evoked Ca²⁺ signaling in human cells, we have investigated the potential of using the sea urchin egg homogenate system for a screening campaign. Known pharmacological inhibitors of NAADP-evoked Ca²⁺ release (but not cADPR- or IP₃-evoked Ca²⁺ release) in this invertebrate system strongly correlated with inhibition of MERS-pseudovirus infectivity in a human cell line. A primary screen of 1534 compounds yielded eighteen ‘hits’ exhibiting >80% inhibition of NAADP-evoked Ca²⁺ release. A validation pipeline for these candidates yielded seven drugs that inhibited NAADP-evoked Ca²⁺ release without depleting acidic Ca²⁺ stores in a human cell line. These candidates displayed a similar penetrance of inhibition in both the sea urchin system and the human cell line, and the extent of inhibition of NAADP-evoked Ca²⁺ signals correlated well with observed inhibition of infectivity of a Middle East Respiratory syndrome coronavirus (MERS-CoV) pseudovirus. These experiments support the potential of this simple, homogenate system for screening campaigns to discover modulators of NAADP, cADPR and IP₃-dependent Ca²⁺ signaling with potential therapeutic value.     

Calcium signalling by nicotinic acid adenine dinucleotide phosphate (NAADP)

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a recently described Ca2+ mobilizing messenger, and probably the most potent. We briefly review its unique properties as a Ca2+ mobilizing agent. We present arguments for its action in targeting acidic calcium stores rather than the endoplasmic reticulum. Finally, we discuss possible biosynthetic pathways for NAADP in cells and candidates for its target Ca2+ release channel, which has eluded identification so far.     

Effect of luminal and extravesicular Ca2+ on NAADP binding and release properties

Nicotinic acid adenine dinucleotide phosphate (NAADP) has been shown to be a powerful Ca2+ release agent in numerous systems, including echinoderms, plants, and mammalian cells. NAADP has been shown to release Ca2+ via a separate mechanism to IP3 and ryanodine receptors, and specific binding sites have recently been characterised. However, functional studies have shown that there is a functional interplay between the NAADP-sensitive mechanism and the other two. In particular, it appears that activation of the NAADP receptor might act as a trigger to facilitate responses from IP3 and ryanodine receptors. To further characterise this interplay, we have investigated the effects of luminal and cytosolic Ca2+ on the NAADP receptor in sea urchin egg homogenates. We report that neither cytosolic nor luminal Ca2+ appears to influence NAADP binding. Conversely, emptying of stores significantly amplifies NAADP-induced fractional Ca2+-release, providing a mechanism of self-adjustment independent of store loading.     

NAADP+ synthesis from cADPRP and nicotinic acid by ADP-ribosyl cyclases

ADP-ribosyl cyclases (ADPRCs) are present from lower Metazoa to mammals and synthesize the Ca2+-active (di)nucleotides cyclic ADP-ribose (cADPR), NAADP+, and ADP-ribose (ADPR), involved in the regulation of important cellular functions. NAADP+ can be synthesized by ADPRCs from NADP+ through a base-exchange reaction, which substitutes nicotinamide for nicotinic acid (NA). Here we demonstrate that ADPRCs from both lower and higher Metazoa (including human CD38) can also synthesize NAADP+ starting from 2'-phospho-cyclic ADP-ribose (cADPRP) and NA. Comparison, on the two substrates cADPRP and NADP+, of the relative rates of the reactions introducing NA and hydrolyzing/cyclizing the substrate, respectively, indicates that with all ADPRCs tested cADPRP is preferentially transformed into NAADP+, while NADP+ is preferentially cyclized or hydrolyzed to cADPRP/2'-phospho-ADP-ribose. cADPRP was detectable in retinoic acid-differentiated, CD38+ HL-60 cells, but not in undifferentiated, CD38- cells. These results suggest that cADPRP may be a NAADP+ precursor in ADPRC+ cells.     

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.     

The Calcium-mobilizing Messenger Nicotinic Acid Adenine Dinucleotide Phosphate Participates in Sperm Activation by Mediating the Acrosome Reaction

Before a sperm can fertilize an egg it must undergo a final activation step induced by the egg termed the acrosome reaction. During the acrosome reaction a lysosome-related organelle, the acrosome, fuses with the plasma membrane to release hydrolytic enzymes and expose an egg-binding protein. Because NAADP (nicotinic acid adenine dinucleotide phosphate) releases Ca²⁺ from acidic lysosome-related organelles in other cell types, we investigated a possible role for NAADP in mediating the acrosome reaction. We report that NAADP binds with high affinity to permeabilized sea urchin sperm. Moreover, we used Mn²⁺ quenching of luminal fura-2 and ⁴⁵Ca²⁺ to directly demonstrate NAADP regulation of a cation channel on the acrosome. Additionally, we show that NAADP synthesis occurs through base exchange and is driven by an increase in Ca²⁺. We propose a new model for acrosome reaction signaling in which Ca²⁺ influx initiated by egg jelly stimulates NAADP synthesis and that this NAADP acts on its receptor/channel on the acrosome to release Ca²⁺ to drive acrosomal exocytosis.     

NAADP as a second messenger: neither CD38 nor base-exchange reaction are necessary for in vivo generation of NAADP in myometrial cells

Nicotinic acid adenine dinucleotide phosphate (NAADP) has recently been shown to act as a second messenger controlling intracellular Ca²⁺ responses in mammalian cells. Many questions remain regarding this signaling pathway, including the role of the ryanodine receptor (RyR) in NAADP-induced Ca²⁺ transients. Furthermore, the exact metabolic pathway responsible for the synthesis of NAADP in vivo has not been determined. Here, we demonstrate that the NAADP mediated Ca²⁺ release system is present in human myometrial cells. We also demonstrate that human myometrial cells use the NAADP second messenger system to generate intracellular Ca²⁺ transients in response to histamine. It has been proposed in the past that the NAADP system in mammalian cells is dependent on the presence of functional RyRs. Here, we observed that the histamine-induced Ca²⁺ transients are dependent on both the NAADP and inositol 1,4,5-trisphosphate signaling pathways but are independent of RyRs. The enzyme CD38 has been shown to catalyze the synthesis of NAADP in vitro by the base-exchange reaction. Furthermore, it has been proposed that this enzyme is responsible for the intracellular generation of NAADP in vivo. Using CD38 knockout mice, we observed that both the basal and histamine stimulated levels of NAADP are independent of CD38 and the base-exchange reaction. Our group is the first to demonstrate that NAADP is a second messenger for histamine-elicited Ca²⁺ transients in human myometrial cells. Furthermore, the NAADP mediated mechanism in mammalian cells can be independent of RyRs and CD38. Our data provides novel insights into the understanding of the mechanism of action and metabolism of this new second messenger system. cADP ribose; inositol 1,4,5-trisphosphate; endoplasmic reticulum; ryanodine channel; nicotinic acid adenine dinucleotide phosphate; CD38; base-exchange reaction     

Sea urchin eggs in the acid reign

Sea urchin eggs have been an indispensable model system for studying egg activation and ionic signalling for at least a century. Instrumental in the discovery of two Ca²⁺-mobilizing second messengers, cyclic ADP-ribose and NAADP, the sea urchin has revolutionized cell biology for all phyla. This review attempts to summarize what we currently know about egg acidic vesicles in the context of Ca²⁺ signalling. The dynamics of Ca²⁺ storage, Ca²⁺ mobilization, proton fluxes and two-pore channels will be discussed.     

Thio-NADP is not an antagonist of NAADP

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a metabolite of NADP, which can release Ca²⁺ from stores that are distinct from those activated by either cyclic ADP-ribose or inositol 1,4,5-trisphosphate (IP₃). It has previously been suggested that thio-NADP is a specific antagonist of NAADP (Chini et al. [1995]J. Biol. Chem. 270, 3216–3223). Its effects in sea-urchin egg homogenates were investigated. At 50 μM, thio-NADP activates partial Ca²⁺ release and totally inhibits subsequent challenge with a saturating concentration of NAADP. Purification by HPLC eliminates the Ca²⁺ releasing activity of 50 μM thio-NADP and reduces the subsequent inhibition by 73.7±1.3%. The residual inhibitory effect is no more than that exerted by 50 μM of either NADP itself or nicotinic acid adenine dinucleotide (NAAD). These results are confirmed by³²P-NAADP binding studies. Unpurified thio-NADP inhibits the specific³²P-NAADP binding to egg microsomes with an IC₅₀ of 40 μM. After HPLC purification, only 20% inhibition is seen at a concentration as high as 50 μM, similar to the extent of inhibition effected by 40 μM NADP. These results indicate the inhibitory substance in thio-NADP is a contaminant. The partial Ca²⁺ release activity of unpurified thio-NADP suggests the contaminant is NAADP itself. This is supported by the fact that pretreatment with a subthreshold concentration of only 2 nM NAADP totally desensitizes the egg homogenates such that no Ca²⁺ response is seen with saturating NAADP. Estimation from the binding studies shows that a contamination of 0.012% of NAADP in the unpurified thio-NADP samples is sufficient to account for the inhibitory effects. These results indicate thio-NADP is not an antagonist of NAADP.     

Molecular Properties of Sodium/Dicarboxylate Cotransporters

Cells possess multiple Ca²⁺ stores and multiple messengers for mobilizing them. In addition to inositol trisphosphate and cyclic ADP-ribose, nicotinic acid adenine dinucleotide phosphate (NAADP), a metabolite of NADP, is shown to be a potent Ca²⁺ signaling molecule in both invertebrate and mammalian cells. This article summarizes the recent results of this newly discovered Ca²⁺ signaling mechanism and explores the implications of the apparent proliferation of Ca²⁺ messengers. Received: 11 August 1999/Revised: 21 September 1999