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

Abstract

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a Ca2؉ -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 NAADPmediated 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 lowaffinity) on the NAADP receptor. Ca2ϩ is an extremely versatile and powerful second messenger (1). Three accepted Ca 2ϩ -releasing second messengers that cells utilize to control the spatiotemporal properties of these Ca 2ϩ signals are inositol 1,4,5-trisphosphate (2), cyclic ADPribose (3), and nicotinic acid adenine dinucleotide phosphate (NAADP) 2 (4). Of these three messengers, little is known about the mechanism of action of NAADP. NAADP is the most potent of the three Ca 2ϩ -releasing messengers (5), and its actions are pharmacologically distinct from inositol 1,4,5-trisphosphate and cyclic ADP-ribose (6 -9). Controversy surrounds both the identity of the NAADP-activated channel and its organellar location. There is evidence for the NAADP receptor being the transient receptor potential mucolipin 1 channel (10), the ryanodine receptor (11, 12), or the two-pore channel (Refs. 13-15; for review, see Ref. 16). There is evidence for the Ca 2ϩ store targeted by NAADP being the endoplasmic reticulum (11) or physically distinct (17), acidic, lysosome-related organelles (18).One of the most intriguing features of NAADP is the relationship between concentration, binding, and response (5,9,19). In sea urchin eggs, a subthreshold concentration of NAADP that does not itself release Ca 2ϩ is able to prevent Ca 2ϩ release in response to a second addition of NAADP that would normally be able to release Ca 2ϩ (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); thus, at low concentrations, NAADP only binds to this site and is able to prevent Ca 2ϩ release via this receptor. In contrast to sea urchin eggs, in mammalian tissue, NAADP has a bell-shaped concentration-r...

Nicotinic acid adenine dinucleotide phosphate regulates skeletal muscle differentiation via action at two-pore channels

“…Both, C2C12 myoblasts and primary murine myoblasts show an increased expression of transcripts that are part of the myogenic differentiation program, such as myogenin and skNAC, when differentiated in the presence of NAADP-AM. On the other hand, both bafilomycin, an inhibitor of the lysosomal H + -ATPase, which is known to inhibit NAADP-dependent calcium release in a number of cell types (33)(34)(35), and Ned-19, a highly selective inhibitor of the NAADP signaling pathway (19,36,37), prevent expression of these transcripts and inhibits the formation of myotubes positive for myosin heavy chain. Taken together, these results are unique in showing that calcium release triggered by NAADP is essential of skeletal muscle differentiation.…”

NAADP as an intracellular messenger regulating lysosomal calcium-release channels

“…Recently, the likely targets for NAADP have been identified as TPCs (two-pore channels), and these have been demonstrated to be distributed in the endolysosomal system, providing direct molecular evidence for NAADP targeting acidic organelles [2]. Another important development has been the characterization of new selective and high-affinity NAADP receptor antagonists, Ned-19 [4] and related compounds [5]. Together, the employment of these new antagonists and the ability to manipulate the molecular targets for NAADP are leading to a better understanding of the mechanism and roles of NAADP as a Ca 2+ -mobilizing messenger, and the establishment of lysosomes and related organelles as messenger-regulated Ca 2+ stores with a crucial role in cellular Ca 2+ signalling [6].…”

Nicotinic Acid Adenine Dinucleotide Phosphate Plays a Critical Role in Naive and Effector Murine T Cells but Not Natural Regulatory T Cells

“…Significant differences between two groups were determined using the unpaired Student's t test. Statistical significance was set at p Ͻ 0.05. release in sea urchin egg homogenates (21,22). CD4 cells were isolated from spleens of untreated C57BL/6 mice.…”