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Published in final edited form as: Biochim Biophys Acta Mol Cell Res. 2018 Dec 4;1866(7):1180–1188. doi: 10.1016/j.bbamcr.2018.11.017

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

Gihan S Gunaratne a, Peiling Su b,1, Jonathan S Marchant c, James T Slama b,1, Timothy F Walseth a,*
PMCID: PMC8101546  NIHMSID: NIHMS1674292  PMID: 30521871

Abstract

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.

Keywords: Nicotinic acid adenine dinucleotide phosphate (NAADP), Clickable NAADP analog, Photoaffinity labeling, Intracellular calcium release, Nucleotide analogs

1. Introduction

Many signal transduction processes converge on fluctuations in cytosolic Ca2+ concentration to regulate a wide assortment of cellular events [1]. Increases in cytosolic Ca2+ concentrations function as a universal signal, which may be, decoded in a remarkably diverse number of ways due to the heterogeneous expression of Ca2+ sensitive effector proteins [1]. Dysfunctional regulation of Ca2+ signals is associated with a large breadth of pathologies, from cardiovascular to neurodegenerative diseases [2]. Therefore, a molecular understanding of the mechanisms governing Ca2+ signaling events underlying these pathologies is important for the informed pursuit of novel therapeutics.

In the context of intracellular Ca2+ signaling, three Ca2+ mobilizing second messengers have thus far been identified. Two of these, inositol triphosphate (IP3) and cyclic-ADP-ribose, induce Ca2+ release from the well-characterized IP3 receptors and ryanodine receptors residing on the endoplasmic reticulum (ER) [3,4]. In contrast, the most recently identified Ca2+ mobilizing messenger, nicotinic acid adenine dinucleotide phosphate (NAADP) [5,6], induces Ca2+ release from “acidic calcium stores”, including endosomes and lysosomes [7,8]. This atypical characteristic is supported by observations that NAADP-evoked Ca2+ release is not inhibited by blockade of ER Ca2+ channels or by depletion of ER Ca2+ stores. NAADP-evoked Ca2+ signals localize to subcellular fractions containing acidic Ca2+ stores but not microsomal stores, and disruption of endolysosomal pH gradient or osmotic rupture of lysosomes abolishes NAADP-sensitivity [7].

Since its discovery, NAADP has been implicated in the signal transduction of a variety of extracellular cues. For example, β-adrenergic receptor signaling stimulates the rapid production of NAADP in cardiac tissues [9,10], where it contributes to the inotropic effects as well hypertrophy and arrhythmias that are characteristically associated with β-adrenergic signaling [11]. Similarly, NAADP-evoked Ca2+ release underlies histamine-induced secretion of von-Willeband Factor in endothelial cells [12] and glucose-induced secretion of insulin in pancreatic beta cells [13], vascular endothelial growth factor-induced neoangiogenesis in mice [14], as well as epidermal growth factor-induced signaling [15]. NAADP contributes to homeostatic cell biology as well, particularly in regard to intracellular trafficking events [16]. NAADP-dependent Ca2+ signaling underpins routine transport of LDL-cholesterol [17], endolysosomal fusion events [18], and autophagy [19,20]. Moreover, NAADP-dependent signaling supports the transport of endocytosed cargo, such as cholera toxin [21] and the cellular entry and infectivity of internalized viral pathogens which traverse the endolysosomal system [22-24]. The contribution of this Ca2+ mobilizing ligand to such a wide spectrum of signaling pathways and biological functions has implicated NAADP in a diversity of pathologies [25]. As such, elucidation of the molecular mechanisms that mediate NAADP signaling is of critical and widespread importance.

The Ca2+ permeable channels that mediate NAADP-evoked Ca2+ release from acidic Ca2+ stores are likely the two-pore channels (TPC1, TPC2) [26,27], though some groups have disputed this notion [28,29], and other candidate channels have been proposed [30,31]. However mounting evidence demonstrates that functional TPC expression is a necessary component of NAADP-signaling. Notably, double-knockout of TPC1 and TPC2 ablated NAADP-evoked Ca2+ signals, while rescue of TPC1 or TPC2 expression restored NAADP-responsiveness [32]. Additionally, a suite of molecular TPC mutants [26,33,34], electro-physiological recordings [35,36], radioligand binding studies [21,27], and overlapping pharmacology [37] support the hypothesis that TPCs are the molecular target of NAADP.

In an effort to characterize NAADP binding sites, previous work by our group employed an unbiased photoaffinity labeling (PAL) approach using a radioactive photoactivatable NAADP analog (32P-5N3-NAADP) [38-40]. In this approach, a photoactivatable azide group is incorporated into the 5-position of the nicotinic ring of NAADP. Upon ultraviolet irradiation, 32P-5N3-NAADP covalently crosslinks to adjacent proteins, and is thus a useful tool for probing for NAADP binding sites. In mammalian systems, photolabeling of NAADP-binding sites revealed a 23 kDa protein with high affinity for 32P-5N3-NAADP, and which was selectively displaced by unlabeled NAADP [38,40]. Pharmacological analysis of the photolabeling of 23 kDa proteins was consistent with properties of NAADP-binding proteins reported from 32P-NAADP radioligand binding studies [41]. Moreover, the pharmacology of the candidate NAADP-binding protein was consistent with the pharmacology of NAADP-evoked Ca2+ release. Subcellular fractionation of SKBR3 cells revealed that the PAL-tagged 23 kDa protein is enriched in soluble fractions (~75%) compared to membrane fractions. Of note, the properties of this soluble 23 kDa NAADP-binding protein suggest that NAADP does not directly associate with TPC1 or TPC2, as immunodetected bands migrated between 90 and 150 kDa (TPC1) and 75–160 kDa (TPC2). Indeed, overexpression or knockout of TPCs did not alter the intensity of labeling of the 23 kDa protein. Similarly, in sea urchin egg homogenates, 32P-5N3-NAADP selectively labeled a 40–45 kDa protein doublet which did not correspond to the molecular weights of immunodetected TPC isoforms, however these labeled NAADP-binding proteins notably co-immunoprecipitated with TPCs [39]. Altogether, these studies suggest that the putative NAADP-binding protein comprises a novel and discrete molecular identity. Identification of the NAADP-binding protein is a critically important step towards understanding the mechanisms governing NAADP-evoked Ca2+ release and downstream cellular processes.

Working towards this goal, we synthesized a bifunctional probe to interrogate the NAADP interactome and identify the NAADP receptor. We previously reported that the 5-position of nicotinic acid and the 8-adenosyl position on NAADP are tolerant to substitution [42,43]. Here, we incorporated a photoactivatable azide group to the 5-position and a “clickable” ethynyl group to the 8-position, yielding the bifunctional NAADP analog, 5N3-8-ethynyl-NAADP. The clickable ethynyl group allows for the addition of a biotin affinity tag via click chemistry [44] for subsequent affinity purification of NAADP-binding proteins. In this study, we aimed to validate 5N3-8-ethynyl-NAADPs ability to engage NAADP-binding proteins by assessing its Ca2+ mobilizing properties and its photoaffinity labeling of putative NAADP-binding proteins in sea urchin and mammalian systems.

2. Results and discussion

2.1. 5-Azido-8-ethynyl-NAADP, a novel bifunctional NAADP analog

A bifunctional NAADP analog was synthesized for the purpose of isolating and identifying NAADP-binding proteins that mediate NAADP-dependent signaling. A photo-activatable azido group was incorporated at the 5- position of the nicotinic acid ring, and a clickable ethynyl moiety was introduced to the 8-adenosyl position of NAADP, yielding 5N3-8-ethynyl-NAADP (Fig. 1). Exposure to ultraviolet (UV) irradiation converts the inert azido group to a nitrene group which initiates covalent crosslinking with local amino acids. Addition of a biotin affinity tag to the 8-ethynyl moiety via click chemistry allows for the selective biotinylation of proteins that associate with 5N3-8-ethynyl-NAADP. The detail of the synthesis and chemical characterization of 5N3-8-ethynyl-NAADP will be published elsewhere.

Fig. 1.

Fig. 1.

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Structure of 5N3-8-ethynyl-NAADP. 5N3-8-ethynyl-NAADP contains a photosensitive azide moiety on the 5-position of the nicotinic acid ring (*) and an ethynyl moiety on the 8-position of the adenine ring (**). The azide moiety allows photocrosslinking of the probe to NAADP binding sites and the ethynyl moiety is a clickable group allowing the addition of biotin through click chemistry.

2.2. 5-Azido-8-ethynyl-NAADP functionally interacts with NAADP receptors

A prerequisite for the utility of a bifunctional probe is that it must retain its affinity for its native receptor(s). To examine whether 5N3-8-ethynyl-NAADP functionally interacts with NAADP receptors, we first tested its efficacy in mobilizing intracellular Ca2+ when microinjected into cultured cells (Fig. 2). For these experiments we employed U2OS cells, a cell line derived from human osteosarcoma, due to their NAADP-responsiveness [22,23] and their morphology which facilitates microinjection. Mobilization of sequestered intracellular Ca2+ was detected by monitoring fluorescence intensity of the genetically encoded calcium indicator, GCaMP6M [45] following microinjection of compounds into single cells. While injection of an intracellular-like buffer was without effect, injection of NAADP (100 nM in pipette) elicited a robust and transient Ca2+ response (peak F/F0 = 4.01 ± 1.2, n = 3 injections). Injection of 1 μM 5N3-8-ethynyl-NAADP induced a small Ca2+ response (peak F/F0 = 2.14 ± 0.37, n = 3). Increasing the pipette concentration of 5N3-8-ethynyl-NAADP to 10 μM resulted in Ca2+ transients of similar magnitude to NAADP (peak F/F0 = 2.8 ± 0.33, n = 3) indicating that the bifunctional analog is less potent than the native NAADP. These data demonstrate that 5N3-8-ethynyl-NAADP is a functional Ca2+ mobilizing messenger in intact human cells.

Fig. 2.

Fig. 2.

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5N3-8-ethynyl-NAADP mobilizes intracellular Ca2+ release in U2OS cells. A., Representative traces of Ca2+ release evoked by microinjection of buffer, NAADP, or 5N3-8-ethynyl-NAADP at the indicated concentrations. B., Quantification of peak ΔF/F0 values from cumulative microinjection datasets, n ≥ 3 injections. C., Pseudocolored images of GCaMP6M fluorescence intensity in response to microinjection of vehicle or compound at the indicated time points. White arrowheads indicate injection sites. Scale bars, 10 μm.

Next, we characterized the ability for 5N3-8-ethynyl to functionally engage the NAADP receptor in sea urchin egg homogenates. In this system NAADP displays atypical concentration-response relationships, in which exposure to sub-threshold concentrations of NAADP that do not evoke Ca2+ responses themselves will in turn inhibit the Ca2+ mobilizing efficacy of subsequent additions NAADP at maximal concentrations in a time- and concentration-dependent manner [46,47]. This ‘self-desensitization’ property is thought to be mediated by a high-affinity inhibitory site on the NAADP receptor, whereas the Ca2+ mobilizing property is thought to be mediated by a lower affinity NAADP-binding site [48,49]. To test 5N3-8-ethynyl-NAADPs functional efficacy in sea urchin egg homogenates, we examined both its ability to desensitize NAADP-responsiveness at lower concentrations, and its ability to elicit Ca2+ responses at higher concentrations. As a control, NAADP was used for comparison. Increasing concentrations of NAADP (5.5 pM–16.5 μM) or 5N3-8-ethynyl-NAADP (15 pM–44 μM) were added to homogenates and fluctuations in free-Ca2+ were monitored using the fluorescent Ca2+ indicator, fluo-3. NAADP displayed a Ca2+ mobilizing EC50 of 7.6 ± 1 nM, in comparison, the bifunctional 5N3-8-ethynyl-NAADP had a calculated EC50 of 2.86 ± 0.4 μM (Fig. 3A). Next, a subsequent addition of 1 μM NAADP was added to all samples, and desensitization to NAADP-evoked Ca2+ release was assessed. The calculated IC50 values for NAADP and 5N3-8-ethynyl-NAADP in this self-desensitization experiment were 0.15 ± 0.008 nM and 99 ± 7.7 nM, respectively.

Fig. 3.

Fig. 3.

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5N3-8-ethynyl-NAADP mobilizes Ca2+ flux and displaces 32P-NAADP in sea urchin egg homogenates. A., Mobilization of sequestered Ca2+ (closed circles, solid lines) and desensitization to subsequent additions of 1 μM NAADP (open circles, dashed lines) in response to the indicated concentrations of NAADP (black) or 5N3-8-ethynyl-NAADP (red), as detected by monitoring peak fluo-3 fluorescence values. Calculated EC50 values for NAADP- and 5N3-8-ethynyl-NAADP-evoked Ca2+ release are 7.6 ± 1 nM and 2.86 ± 0.4 μM, respectively. Calculated IC50 values for NAADP- and 5N3-8-ethynyl-NAADP- induced desensitization to 1 μM NAADP stimulus are 0.15 ± 0.008 nM and 99 ± 7.7 nM, respectively. Data represents averaged results of n = 3 experiments, ± STDEV. B., Radioligand competition binding assays. Sea urchin egg homogenates were incubated with 32P-NAADP (0.15 nM) in the presence of the indicated concentration of unlabeled NAADP (black) or 5N3-8-ethynyl-NAADP (red), total bound radiolabel was quantified by scintillation counting. Data represents averaged results of n = 3 experiments, ± STDEV.

As an additional method for validating the capacity of 5N3-8-ethynyl-NAADP to engage NAADP-binding proteins, we conducted radioligand competition binding assays in sea urchin egg homogenates. This approach allows for the direct measurement of 5N3-8-ethynyl-NAADPs ability to compete with radiolabeled [32P]-NAADP for the same binding sites. Homogenate was incubated with [32P]-NAADP (0.15 nM) in the presence of increasing concentrations of NAADP or 5N3-8-ethynyl-NAADP for 75 min to allow receptor-ligand interactions to achieve an equilibrium. After incubation, homogenates were filtered, free-radiolabel removed by washing, and total bound [32P]-NAADP was quantified by scintillation counting. Displacement of bound [32P]-NAADP by unlabeled NAADP (IC50 = 0.26 ± 0.02 nM) was several orders of magnitude more potent than displacement by 5N3-8-ethynyl-NAADP (IC50 = 123 ± 26 nM) (Fig. 3B). Encouragingly, the relative potency of 5N3-8-ethynyl-NAADP compared to the native NAADP is comparable between Ca2+ release assays, self-desensitization assays, and radioligand binding assays. Collectively, these data demonstrate that 5N3-8-ethynyl-NAADP is a low-potency Ca2+ mobilizing agonist that binds the NAADP-receptor in sea urchin egg homogenate.

2.3. Validation of photolabeling of putative NAADP-binding proteins using [32P]-5N3-8-ethynyl-NAADP

We then employed a radiolabeled analog of 5N3-8-ethynyl-NAADP to test if this bifunctional probe labeled the same proteins that we previously characterized using the monofunctional photoprobe, [32P]-5N3-NAADP. In our earlier studies in SKBR3, HEK293 and Jurkat cells and mouse pancreas [38,40], we reported that putative NAADP-binding proteins are enriched in soluble fractions in mammalian systems. For this reason, in the present study, soluble fractions of U2OS and Jurkat cell extracts were used for PAL experiments. U2OS cell extracts were incubated with either [32P]-5N3-NAADP or [32P]-5N3-8-ethynyl-NAADP and exposed to UV irradiation, and samples were separated by electrophoresis, resulting in the identification of multiple labeled bands of various molecular masses (Fig. 4A). Importantly, control samples lacking UV irradiation displayed no photolabeling due to lack of protein-ligand crosslinking. Incubation of either photoprobe in the presence of unlabeled NAADP, to shield NAADP-binding sites, prevented photolabeling of two bands with approximate molecular masses of 35 kDa (*) and 23 kDa (**) (Fig. 4A). Labeled bands that are not displaced by the presence of unlabeled NAADP represents non-specific labeling. Inspection of the distribution of labeled bands suggests that both the monofunctional and the bifunctional photoprobes largely interact with the same collection of proteins, though the intensity of labeled bands is variable. The relative affinity of either photoprobe for the two selectively labeled proteins was assessed by densitometry analysis (Fig. 4B). The labeling of both the 35 kDa protein and the 23 kDa protein was approximately 3-fold higher in samples incubated with [32P]-5N3-NAADP versus [32P]-5N3-8-ethynyl-NAADP. Incubating low concentrations of radiolabeled [32P]-5N3-8-ethynyl-NAADP (< 1 nM) with sea urchin egg homogenates failed to selectively label any putative NAADP-binding proteins, in contrast to [32P]-5N3-NAADP which displayed selective labeling of a 40–45 kDa doublet and a 30 kDa band, consistent with previous studies [39]. The higher affinity of 5N3-8-ethynyl-NAADP for putative NAADP-binding proteins in mammalian samples relative to sea urchin samples is consistent with the Ca2+ mobilizing properties described above (Fig. 2, Fig. 3). These data demonstrate that low concentrations of [32P]-5N3-8-ethynyl-NAADP are capable of recognizing and selectively labeled NAADP binding proteins in mammalian samples. However we were unable to achieve the higher concentrations of radioligand that are likely necessary for labeling sea urchin NAADP binding proteins.

Fig. 4.

Fig. 4.

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Comparison of [32P]-5N3-NAADP and [32P]-5N3-8-ethynyl-NAADP Photoaffinity Labeling. Phosphorimages of photolabeled U2OS cell extracts (A) or sea urchin egg homogenates (C). A., U2OS extracts were labeled with either [32P]-5N3-NAADP (0.57 nM) or [32P]-5N3-8-ethynyl-NAADP (0.57 nM) in the presence of increasing concentrations of NAADP, and separated by SDS-PAGE. Bands that are selectively displaced by NAADP are noted, 35 kDa proteins (*), 23 kDa proteins (**). B., Densitometry analysis of photolabeling of 23 kDa proteins and 35 kDa proteins by [32P]-5N3-NAADP (black bars) or [32P]-5N3-8-ethynyl-NAADP (red bars). C., Sea urchin egg homogenates were labeled with either [32P]-5N3-NAADP (0.24 nM) or [32P]-5N3-8-ethynyl-NAADP (0.23 nM) in the absence or presence of unlabeled NAADP (1 μM) and separated by SDS-PAGE. Data is shown is a representative experiment (n = 3).

In order to characterize the kinetics of the association between [32P]-5N3-8-ethynyl-NAADP and target proteins was assessed by conducting time-course PAL experiments (Fig. 5). Samples were incubated for the indicated times with [32P]-5N3-8-ethynyl-NAADP in the dark and then exposed to UV for 2 min. UV irradiation after a brief 20 s incubation with U2OS cell extracts resulted in rapid labeling of both the 23 kDa protein and the 35 kDa protein. This effect was time-dependent, as both proteins exhibited increased photolabeling over the course of 60 min (Fig. 5). Next, we sought to test for the presence of low affinity NAADP-binding proteins by incubating U2OS cell extracts with increasing concentrations of [32P]-5N3-8-ethynyl-NAADP (0.014 nM–4.7 nM) (Fig. 6). Increasing concentrations of photoprobe resulted in more intense labeling of previously identified bands, but failed to reveal any novel potential NAADP-binding proteins (Fig. 6).

Fig. 5.

Fig. 5.

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Time course of [32P]-5N3-8-ethynyl-NAADP photolabeling. A., Phosphorimage of U2OS cell extracts incubated with 0.54 nM [32P]-5N3-8-ethynyl-NAADP for the indicated duration prior to ultraviolet irradiation and separated by SDS-PAGE. Control sample lacking ultraviolet irradiation (No UV) was incubated for 30 min in the presence of photoprobe. B., Densitometry analysis of total labeling of 23 kDa proteins (black symbols and line) and 35 kDa protein (red symbols and line) over time (n = 1).

Fig. 6.

Fig. 6.

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Photolabeling with increasing concentrations of [32P]-5N3-8-ethynyl-NAADP. A., Phosphorimage of U2OS cell extracts labeled with the indicated concentration of [32P]-5N3-8-ethynyl-NAADP for 60 min in the absence or presence of unlabeled NAADP (1 μM) and separated by SDS-PAGE. Bands that are selectively displaced by NAADP are noted, 35 kDa proteins (*), 23 kDa proteins (**). B., Densitometry analysis of specific labeling of 23 kDa proteins (n = 1).

Finally, a more detailed characterization of the selectivity of the 23 kDa and 35 kDa proteins was conducted. To affirm the selectivity of these putative NAADP-binding proteins, we incubated U2OS cell extracts with [32P]-5N3-8-ethynyl-NAADP and increasing concentrations of NAADP or the structurally related NADP (Fig. 7). Displacement of photolabeling of the 23 kDa protein was ~9-fold more penetrant with NAADP (IC50 = 27 ± 1.3 nM) compared to NADP (IC50 = 246 ± 13nM) (Fig. 5B-C). In contrast to the 23 kDa band, pharmacological assessment of concentration-dependent displacement of photolabeling of the 35 kDa band demonstrated a ~8-fold lower affinity for NAADP (IC50 = 213 ± 19 nM) as well as less selectivity relative to NADP (IC50 = 940 ± 204 nM) (Fig. 7B-C). Jurkat cell extracts were employed in a parallel PAL experiment. Reassuringly, densitometry analysis of selective displacement of [32P]-5N3-8-ethynyl-NAADP in this additional human cell line was in strong agreement with properties found in U2OS cell extracts (Fig. 7B-C).

Fig. 7.

Fig. 7.

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Selectivity of [32P]-5N3-8-ethynyl-NAADP photolabeling. A., Phosphorimage of U2OS cell extracts incubated with [32P]-5N3-8-ethynyl-NAADP (0.23 nM) in the presence of the indicated concentrations of NAADP or NADP, and separated by SDS-PAGE. Bands that are selectively displaced by NAADP are noted, 35 kDa proteins (*), 23 kDa proteins (**). B. Densitometry analysis of displacement of photolabeling of 23 kDa proteins (closed circles, solid lines) and 35 kDa proteins (open circles, dashed lines) with NAADP (black) or NADP (red) in U2OS cell extracts (left) or Jurkat cell extracts (right). C. Comparison of calculated IC50 values for displacement of 23 kDa proteins (top) or 35 kDa proteins (bottom) by NAADP (black) or NADP (red). Phosphorimage of Jurkat extract labeling is not shown. Data shown is a representative experiment (n = 2).

Together, these PAL data demonstrate that 5N3-8-ethynyl-NAADP rapidly and selectively associates with NAADP-binding proteins. The ability for this bifunctional probe to covalently link to NAADP-binding proteins, coupled with the amenability of the ethynyl group for the addition of biotin affinity tags via click chemistry, potentially make 5N3-8-ethynyl-NAADP a powerful probe for future isolation of the elusive NAADP receptor(s).

3. Methods

3.1. Plasmids

Plasmid encoding GCaMP6M was a gift from Douglas Kim (Addgene plasmid #40754).

3.2. Cell culture and transfection

U2OS cells were maintained in DMEM, Jurkat cells were maintained in RPMI medium. Cell culture media was supplemented with 10% fetal bovine serum, 100 units/ml penicillin and streptomycin and 292 μg/ml l-glutamine, and cells were cultured at 5% CO2 and 37 °C. For transfection, 4 × 105 U2OS cells were seeded in a 35 mm well in culture media without antibiotics. Transfection complexes were added before cells adhered to the culture surface, using Lipofectamine LTX with PLUS reagent and 2.5μg DNA, according the vendor's recommendation. After 24 h, cells were trypsinized and seeded into imaging dishes. Jurkat cells were purchased from ATCC. U2OS cells were a gift from Eugen Brailoiu. All cell culture reagents were purchased from Thermo Fisher.

3.3. Preparation of cell extracts

U2OS cells were collected by scraping. U2OS and Jurkat cells were rinsed in PBS and pelleted at 800RCF/5 min three times. Cell pellets were resuspended in 20 mM HEPES supplemented with EDTA-free protease and phosphatase inhibitor cocktail (Thermo Fisher), pH 7.4. Cells were lysed by sonication at 20 kHz with three 15 s pulses. Cell debris was pelleted by centrifuging at 1000xRCF for 5 min, supernatant (S1) was transferred to ultracentrifuge tubes. S1 supernatant was centrifuged at 200,000xRCF for 90 min at 4C in a Beckman Optima XPN 100 ultracentrifuge, using an SW-55 Ti rotor. Supernatant (S200) was collected, protein was quantified by Bradford assay, and samples were aliquotted and frozen at −80 °C.

3.4. Confocal Ca2+ imaging and microinjection

U2OS cells were transfected with plasmid encoding GCaMP6M and seeded onto collagen-coated 35 mm glass-bottom dishes (MatTek). Dishes were mounted on an Olympus IX81 microscope for imaging. Cells were perfused with Ca2+-free Hank's Balanced Salt Solution (Thermo Fisher) at a rate of 0.5 ml/min. Isolated cells expressing GCaMP6M were identified by fluorescence. Cell morphology was assessed by acquiring z-stack images and reconstructing three-dimensional models of each cell to be injected. Regions that were not relatively close to either the nucleus or the cell periphery were targeted for injection sites. Femtotip (Eppendorf) injection pipettes were backfilled with intracellular buffer (110 mM KCl, 10 mM NaCl, 20 mM HEPES, pH 7.2) containing either vehicle or drug, and were positioned using an Injectman-4 (Eppendorf) micromanipulation system. Cells were injected at a z-position that was approximately 70% of the cell thickness at the site of injection using a Femtojet4i (Eppendorf). Injection parameters were 85 hPa injection pressure, 40 hPa compensation pressure, 0.5 s injection duration, 45° injection angle, and 600 μm/s injection speed. Cells to be injected were imaged (λex 488 nm, λem 514 ± 15 nm bandpass) using a Plan-Apochromat 60×/1.42 oil differential interference contrast objective, and fluorescence changes were monitored using a Yokogawa spinning disk confocal (CSU-X-M1N), and an Andor iXon Ultra 888 EMCCD camera. Image acquisition and data collection was done using Metamorph version 7.10.

3.5. Synthesis of 8-ethynyl-NAD, 8-ethynyl-NADP and 5N3-NAADP

The details of the synthesis and chemical characterization of 8-ethynyl-substituted analogs will be published elsewhere.

3.6. Ca2+ release, NAADP desensitization and radioligand binding assays in sea urchin egg homogenate

Strongylocentrotus purpuratus egg homogenates preparations, Ca2+ release assays, sub-threshhold desensitization of NAADP responsiveness, and [32P]-NAADP competition binding were all performed as previously described [43].

3.7. Synthesis of [32P]-NAADP, [32P]-5N3-NAADP and [32P]-5N3-8-ethynyl-NAADP

The [32P]-labeled probes were synthesized using a two-step strategy used previously [39]. In the first step, [32P-gamma]-ATP (6000 Ci/mmol) was used to phosphorylate NAD or 8-ethynyl-NAD at the 2′-position using recombinant human NAD kinase. The resulting [32P]-NADP or [32P]-8-ethynyl-NADP were converted to the appropriate probe through a base-exchange reaction catalyzed by recombinant Aplysia ADP-riboysl cyclase [50]. The conditions for each base-exchange reaction for the production of each probe are detailed below. The [32P]-NADP or [32P]-8-ethynyl-NADP were dried in a Savant SpeedVac concentrator prior to the base-exchange reactions.

[32P]-NAADP was produced by adding 200 μl of 50 mM nicotinic acid in 100 mM sodium acetate, pH 4 to a vial of dried [32P]-NADP. The reaction was started by adding 1 μl of 0.2 mg/ml wild type Aplysia ADP-ribosyl cyclase and allowed to incubate for 2 h at room temperature in the dark.

[32P]-5N3-NAADP was produced by adding 100 μl of 40 mM 5-azido-nicotinic acid, pH 4 to a vial of dried [32P]-NADP. The reaction was started by adding 1 μl of 0.2 mg/ml Aplysia ADP-ribosyl cyclase and allowed to incubate for 5 h at room temperature in the dark.

[32P]-5N3-8-ethynyl-NAADP was produced by adding 100 μl of 40 mM 5-azido-nicotinic acid, pH 4 to a dried aliquot of [32P]-8-ethynyl-NADP. The reaction was started by adding 1 μl of 0.2 mg/ml E98 Aplysia ADP-ribosyl cyclase and allowed to incubate at room temperature in the dark for 5 h. All reactions were purified by chromatography on a AG MP-1 column as previously described [39].

3.8. Photoaffinity labeling studies

The conditions for photoaffinity labeling of sea urchin egg homogenates and mammalian cell extracts have been previously described [38-40]. The standard condition utilized 0.1–1.5 nM 32P-labeled probe (5N3-NAADP or 5N3-8-ethynyl-NAADP) and 30–50 μg protein (mammalian cell extract or sea urchin egg homogenate). The samples were incubated with the photoprobes for 60 min at 4 °C in the dark and then subjected to UV irradiation for 2 min in a Raynet UV chamber. The photoprobes were used at the same concentrations in experiments where the labeling with 32P-5N3-NAADP was compared to 32P-5N3-8-ethynyl-NAADP (see Figure legends for details). The photolabeled samples were separated by SDS-PAGE on 12% TGX gels from Bio-Rad. The resulting gels were stained with Simply Blue Safestain (Thermo Fisher) and air-dried between cellophane sheets. The photolabeling was analyzed by exposing the dried gels to MP storage phosphor screens (Packard Instruments). The screens were developed using a Typhoon storage phosphor system. Densitometric analysis was accomplished using Image J software.

Acknowledgements

J.T.S. and T.F.W. acknowledge financial support from NIH Institute of General Medical Sciences Grant Number GM100444. J.S.M and G.S.G. received support from NIH GM088790. Work at the University of Toledo was partially supported by The University of Toledo Foundation Cancer Research Support Account.

Abbreviations

NAADP

nicotinic acid adenine dinucleotide 2′-phosphate

PAL

photoaffinity labeling

5N3

5-azido

LDL

low density lipoprotein

UV

ultraviolet

NADP

nicotinamide adenine dinucleotide 2′-phosphate

Footnotes

☆

This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.

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