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

Daniel Rosen,Alexander M Lewis,Akiko Mizote,Justyn M Thomas,Parvinder K Aley,Sridhar R Vasudevan,Raman Parkesh,Antony Galione,Minoru Izumi,A Ganesan,Grant C Churchill

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.

Highlights

One of the most intriguing features of Nicotinic acid adenine dinucleotide phosphate (NAADP) is the relationship between concentration, binding, and response (5, 9, 19)
A subthreshold concentration of NAADP that does not itself release Ca2ϩ is able to prevent Ca2ϩ release in response to a second addition of NAADP that would normally be able to release Ca2ϩ (20, 21). This unusual desensitization may relate to the formation of a spatial and temporal “memory” (22, 23). This effect is thought to be due to a second high-affinity inhibitory site present on the receptor (24); at low concentrations, NAADP only binds to this site and is able to prevent Ca2ϩ release via this receptor
To meet the need for selective and useful chemical probes for studying NAADP signaling, we have synthesized a cell-permeant form of NAADP, NAADP-acetoxymethyl ester, which is cleaved by esterases intracellularly to become biologically active (28), and identified a drug-like, cell-permeant, and selective NAADP antagonist, Ned-19 (29)

Summary

Introduction

One of the most intriguing features of NAADP is the relationship between concentration, binding, and response (5, 9, 19). This effect is thought to be due to a second high-affinity inhibitory site present on the receptor (24); at low concentrations, NAADP only binds to this site and is able to prevent Ca2ϩ release via this receptor. We used the sea urchin homogenate bioassay to assess the activity of the Ned-19 analogues on NAADP-mediated Ca2ϩ release (31).

Results

Conclusion