title: Photoaffinity labeling of nicotinic acid adenine dinucleotide phosphate (NAADP) targets in mammalian cells.
abstract: Nicotinic acid adenine dinucleotide phosphate (NAADP) is an agonist-generated second messenger that releases Ca(2+) from intracellular acidic Ca(2+) stores. Recent evidence has identified the two-pore channels (TPCs) within the endolysosomal system as NAADP-regulated Ca(2+) channels that release organellar Ca(2+) in response to NAADP. However, little is known about the mechanism coupling NAADP binding to calcium release. To identify the NAADP binding site, we employed a photoaffinity labeling method using a radioactive photoprobe based on 5-azido-NAADP ([(32)P-5N(3)]NAADP) that exhibits high affinity binding to NAADP receptors. In several systems that are widely used for studying NAADP-evoked Ca(2+) signaling, including sea urchin eggs, human cell lines (HEK293, SKBR3), and mouse pancreas, 5N(3)-NAADP selectively labeled low molecular weight sites that exhibited the diagnostic pharmacology of NAADP-sensitive Ca(2+) release. Surprisingly, we were unable to demonstrate labeling of endogenous, or overexpressed, TPCs. Furthermore, labeling of high affinity NAADP binding sites was preserved in pancreatic samples from TPC1 and TPC2 knock-out mice. These photolabeling data suggest that an accessory component within a larger TPC complex is responsible for binding NAADP that is unique from the core channel itself. This observation necessitates critical evaluation of current models of NAADP-triggered activation of the TPC family.
authors: Department of Pharmacology, University of Minnesota Medical School, Minneapolis, Minnesota 55455, USA.
authors: Yaping
authors: Y
authors: Lin-Moshier
authors: Timothy F
authors: TF
authors: Walseth
authors: Dev
authors: D
authors: Churamani
authors: Sean M
authors: SM
authors: Davidson
authors: James T
authors: JT
authors: Slama
authors: Robert
authors: R
authors: Hooper
authors: Eugen
authors: E
authors: Brailoiu
authors: Sandip
authors: S
authors: Patel
authors: Jonathan S
authors: JS
authors: Marchant
mesh_terms: D000818:Animals; D001665:Binding Sites; D015220:Calcium Channels / Q000378:metabolism*; D020013:Calcium Signaling / Q000502:physiology*; D045744:Cell Line, Tumor; D057809:HEK293 Cells; D006801:Humans; D015640:Ion Channel Gating / Q000502:physiology*; D051379:Mice; D018345:Mice, Knockout; D009249:NADP / Q000031:analogs & derivatives* / Q000378:metabolism; D010179:Pancreas / Q000378:metabolism*; D019920:Photoaffinity Labels / Q000737:chemistry; D000097903:Two-Pore Channels
chemical_list: D015220:Calcium Channels; D009249:NADP; D019920:Photoaffinity Labels; C407357:TPCN1 protein, human; D000097903:Two-Pore Channels; C530224:TPCN2 protein, human; C024376:NAADP