Analogues of the nicotinic acid adenine dinucleotide phosphate (NAADP) antagonist Ned-19 indicate two binding sites on the NAADP receptor.
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.
Rosen
Daniel
D
Department of Pharmacology, University of Oxford, Mansfield Road, Oxford OX1 3QT, United Kingdom.
Lewis
Alexander M
AM
Mizote
Akiko
A
Thomas
Justyn M
JM
Aley
Parvinder K
PK
Vasudevan
Sridhar R
SR
Parkesh
Raman
R
Galione
Antony
A
Izumi
Minoru
M
Ganesan
A
A
Churchill
Grant C
GC
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
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