Lin-Moshier, Y; Walseth, TF; Churamani, D; Davidson, SM; Slama, JT; Hooper, R; ... Marchant, JS; + view all Lin-Moshier, Y; Walseth, TF; Churamani, D; Davidson, SM; Slama, JT; Hooper, R; Brailoiu, E; Patel, S; Marchant, JS; - view fewer (2012) Photoaffinity labeling of nicotinic acid adenine dinucleotide phosphate (NAADP) targets in mammalian cells. J Biol Chem , 287 (4) 2296 - 2307. 10.1074/jbc.M111.305813.
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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.
|Title:||Photoaffinity labeling of nicotinic acid adenine dinucleotide phosphate (NAADP) targets in mammalian cells.|
|Keywords:||Animals, Binding Sites, Calcium Channels, Calcium Signaling, Cell Line, Tumor, HEK293 Cells, Humans, Ion Channel Gating, Mice, Mice, Knockout, NADP, Pancreas, Photoaffinity Labels|
|UCL classification:||UCL > School of Life and Medical Sciences > Faculty of Life Sciences > Biosciences (Division of) > Cell and Developmental Biology|
UCL > School of Life and Medical Sciences > Faculty of Population Health Sciences > Institute of Cardiovascular Science
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