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 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.

FIGURE 1.

Structural comparisons of NAADP and the Ned-19 analogues. A, two-dimensional chemical structure of NAADP, Ned-19, Ned-19.4, and Ned-20. Structural groups that mimic one another are highlighted in the same color. B, three-dimensional overlay of NAADP (black) and Ned-19.4 (red) illustrating the basis of the mimicry.

FIGURE 2.

Ned-19 analogues exhibit different effects on NAADP-induced Ca²⁺ release and [³²P]NAADP binding indicating two binding sites for NAADP on its receptor. A, effects of Ned-19 analogue concentration on NAADP-induced Ca²⁺ release. Sea urchin egg homogenate was preincubated (3–5 min) with the indicated concentrations of Ned-19 analogues. The Ca²⁺ response to 50 nm NAADP (approximate EC₅₀) was then measured. B, Ned-19.4 selectively inhibits NAADP-mediated Ca²⁺ release. Ca²⁺ release mediated by ionomycin (5 μm), inositol 1,4,5-trisphosphate (IP₃; 5 μm), and cyclic ADP-ribose (cADPR; 1 μm) is unaffected by Ned-19.4 compared with the control (1% DMSO). AFU, arbitrary fluorescence units. C, effect of several Ned-19.4 concentrations on the concentration-response relationship for NAADP-induced Ca²⁺ release. The control is labeled “0” and contained 1% DMSO. For NAADP, the EC₅₀ values are as follows: 65 nm (control), 75 nm (100 μm Ned-19.4), and 65 nm (30 μm Ned-19.4). D, effect of NAADP and Ned-19 analogues on [³²P]NAADP binding. The IC₅₀ values are as follows: Ned-19, 4 μm; Ned-20, 1 μm; Ned-19.4 does not bind.

FIGURE 3.

Ned-20 prevents NAADP self-desensitization. A, example trace of a control response to 100 nm NAADP following a 5-min incubation with vehicle (1% DMSO). B, example trace of a desensitization response to a low concentration of NAADP. Sea urchin egg homogenate was incubated with a low concentration of NAADP (1 nm) for 5 min before the addition of 100 nm NAADP. C, example trace showing Ned-20-mediated inhibition of the NAADP self-desensitization response. Preincubation (10 min) with Ned-20 (100 μm) prevented self-desensitization induced by 1 nm NAADP. D, effect of Ned-20 concentration on inhibition of the NAADP self-desensitization response. The experiment was conducted as in C but with the concentration of Ned-20 varied. RFU, relative fluorescence units.

FIGURE 4.

Model for the action of NAADP at its receptor in sea urchin egg. A, schematic depicting the NAADP receptor channel illustrating a possible relationship between receptor state (function) and orthosteric and allosteric binding sites. B, mechanistic three-state model for the NAADP receptor. The receptor is in thermodynamic equilibrium with all three states, with the most prominent state being “resting” in the absence of ligand. High concentrations of NAADP bind to the low-affinity orthosteric site of the open receptor state, stabilizing this conformation and shifting the equilibria of the states to “open,” resulting in Ca²⁺ release. The orthosteric binding site can be antagonized by Ned-19.4. Low concentrations of NAADP bind to the high-affinity allosteric site on the “locked” receptor state, stabilizing this state and thereby shifting the equilibria and resulting in self-desensitization. Binding to the allosteric site can be detected with [³²P]NAADP. Either the bound NAADP at the allosteric site is occluded, or the off rate is slow enough to make binding effectively irreversible over the time course of the experiments. This allosteric binding site activity can be reversibly antagonized by Ned-20.