Analogues of the nicotinic acid adenine dinucleotide phosphate (NAADP) antagonist ned-19 indicate two binding sites on the NAADP receptor

Rosen, Daniel, Lewis, Alexander M., Mizote, Akiko, Thomas, Justyn M., Aley, Parvinder K., Vasudevan, Sridhar R., Parkesh, Raman, Galione, Anthony, Izumi, Minoru, Ganesan, A. and Churchill, Grant C. (2009) Analogues of the nicotinic acid adenine dinucleotide phosphate (NAADP) antagonist ned-19 indicate two binding sites on the NAADP receptor. The Journal of Biological Chemistry, 284 (50), 34930-34934. (doi:10.1074/jbc.M109.016519).

Abstract

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a Ca(2+)-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 Ca(2+) release and NAADP binding.

A fluorometry bioassay was used to assess NAADP-mediated Ca(2+) 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 Ca(2+) release but inhibits NAADP binding. Conversely, Ned-19.4 (a methyl ester of Ned-19) inhibits NAADP-mediated Ca(2+) 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.

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