Triazine dyes are agonists of the NAADP receptor

Abstract

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. 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+) release and be used for affinity chromatography of the receptor. 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. The compound that displayed the highest affinity, RR120, was tested in a (45)Ca(2+) efflux assay. This compound released Ca(2+) 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+) release. 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. 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.

Figure 1

Binding curves for triazine dyes competing with [³²P]NAADP in sea urchin egg homogenate. n=9–12.

Figure 2

⁴⁵Ca²⁺ release in response to NAADP (2 μM) and RR120 (100 μM) (filled bars). Open bars show the release following pretreatment with 3 nM NAADP. n=9–14.

Figure 3

(a) Inhibition of NAADP receptor binding to RG19 agarose by increasing concentrations of NaCl. n=6–9. (b) Inhibition of NAADP receptor binding to RG5 agarose by BaCl₂ and NaCl. n=8–9. (c) Partial purification of the NAADP receptor using a RG5 agarose column. The relative protein content of each fraction (% of initial 1 mg solubilised protein applied to column) was measured by Bradford assay. Specific [³²P]NAADP binding detected in each fraction is expressed as the % of total [³²P]NAADP binding detected in 1 mg solubilised protein. n=6.