title: 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.
authors.affiliation: Department of Pharmacology, University of Oxford, Mansfield Road, Oxford OX1 3QT, United Kingdom.
authors.forename: Daniel
authors.initials: D
authors.lastname: Rosen
authors.forename: Alexander M
authors.initials: AM
authors.lastname: Lewis
authors.forename: Akiko
authors.initials: A
authors.lastname: Mizote
authors.forename: Justyn M
authors.initials: JM
authors.lastname: Thomas
authors.forename: Parvinder K
authors.initials: PK
authors.lastname: Aley
authors.forename: Sridhar R
authors.initials: SR
authors.lastname: Vasudevan
authors.forename: Raman
authors.initials: R
authors.lastname: Parkesh
authors.forename: Antony
authors.initials: A
authors.lastname: Galione
authors.forename: Minoru
authors.initials: M
authors.lastname: Izumi
authors.forename: A
authors.initials: A
authors.lastname: Ganesan
authors.forename: Grant C
authors.initials: GC
authors.lastname: Churchill
mesh_terms: D000818:Animals; D001665:Binding Sites; D001681:Biological Assay / Q000379:methods; D002118:Calcium / Q000378:metabolism; D002243:Carbolines / Q000737:chemistry / Q000378:metabolism*; D015394:Molecular Structure; D009249:NADP / Q000031:analogs & derivatives* / Q000037:antagonists & inhibitors; D009865:Oocytes / Q000166:cytology / Q000378:metabolism; D010879:Piperazines / Q000737:chemistry / Q000378:metabolism*; D011869:Radioligand Assay; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism*; D012617:Sea Urchins
chemical_list: C539827:1-(3-((4-(2-fluorophenyl)piperazin-1-yl)methyl)-4-methoxyphenyl)-2,3,4,9-tetrahydro-1H-pyrido(3,4-b)indole-3-carboxylic acid; D002243:Carbolines; D010879:Piperazines; D011956:Receptors, Cell Surface; D009249:NADP; C024376:NAADP; D002118:Calcium