Triazine dyes are agonists of the NAADP receptor

Abstract

1 NAADP has been shown to be a potent calcium-releasing second messenger in a wide variety of cell types to date. However, research has been hampered by a lack of pharmacological agents, with which to investigate NAADP-induced calcium release, and by the molecular identity of its cellular target protein being unknown. 2 In the present paper, the sea urchin egg model was used to investigate whether triazine dyes, which can act as nucleotide mimetics, can bind to the NAADP receptor, induce Ca 2 þ release and be used for affinity chromatography of the receptor. 3 Indeed, all the triazine dyes tested (Reactive Red 120 (RR120), Reactive Green 19 (RG19), Reactive Green 5 (RG5), Cibacron Blue 3GA and Reactive Yellow 86) displayed micromolar affinities, except for Reactive Orange 14. Furthermore, unlike NAADP, RR120, RG19 and RG5 did not bind in an irreversible manner. 4 The compound that displayed the highest affinity, RR120, was tested in a 45 Ca 2 þ efflux assay. This compound released Ca 2 þ via the NAADP receptor, as shown by the ability of subthreshold NAADP concentrations to inhibit this release. Furthermore, heparin and ruthenium red were unable to block RR120-induced Ca 2 þ release. 5 We have also shown that RG5 and RG19, immobilised on resins, retain the ability to bind to the receptor, and that this interaction can be disrupted by high salt concentrations. As a proof of principle, we have shown that this can be used to partially purify the NAADP receptor by at least 75-fold. 6 In conclusion, triazine dyes interact with the NAADP receptor, and this could be exploited in future to create a new generation of pharmacological tools to investigate this messenger and, in combination with other techniques, to purify the receptor.

The Molecular Basis for Ca<SUP>2</SUP><SUP>+</SUP> Signalling by NAADP: Two-Pore Channels in a Complex?

“…Soluble preparations bind NAADP with similar potency and selectivity as membranes (Berridge et al, 2002). Substantial enrichment can be obtained using affinity columns based on triazine dyes which act as NAADP agonists (Billington et al, 2004). The non-dissociating nature of NAADP binding allowed convenient tracking during several biochemical fractionation procedures and definition of key properties of the ligand-bound target.…”

Two-pore channels: Regulation by NAADP and customized roles in triggering calcium signals

“…Again, unusually, the calculated native molecular weight differed depending on the fractionation method [23]. Attempts to purify the target protein to homogeneity have been conspicuously unsuccessful although Billington et al did achieve substantial enrichment following a one step fractionation [24]. Taken together, multiple lines of independent evidence suggested that NAADP targeted a novel calcium-permeable channel.…”

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

“…Homogenate can be prepared in bulk, and stored as frozen aliquots which remain responsive for many years. For all these reasons, the system has long been regarded as the ‘gold-standard’ for studying NAADP action [ 34 , 35 ], and has been frequently used to assess the action of molecules eliciting Ca 2+ release through each of the discrete, endogenous Ca 2+ mobilization pathways [ 13 , 30 , 32 , 33 , [37] , [38] , [39] , [40] , [41] , [42] ]. Although the system is clearly amenable to high throughput profiling, no screening campaign has, to our knowledge, yet been reported.…”