Photoaffinity labeling of nicotinic acid adenine dinucleotide phosphate (NAADP) targets in mammalian cells. J Biol Chem 2012 Jan 20;287(4):2296-307
Date
11/26/2011Pubmed ID
22117075Pubmed Central ID
PMC3268391DOI
10.1074/jbc.M111.305813Scopus ID
2-s2.0-84856068326 (requires institutional sign-in at Scopus site) 141 CitationsAbstract
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.
Author List
Lin-Moshier Y, Walseth TF, Churamani D, Davidson SM, Slama JT, Hooper R, Brailoiu E, Patel S, Marchant JSAuthor
Jonathan S. Marchant PhD Chair, Professor in the Cell Biology, Neurobiology and Anatomy department at Medical College of WisconsinMESH terms used to index this publication - Major topics in bold
AnimalsBinding Sites
Calcium Channels
Calcium Signaling
Cell Line, Tumor
HEK293 Cells
Humans
Ion Channel Gating
Mice
Mice, Knockout
NADP
Pancreas
Photoaffinity Labels