Photoaffinity labeling of nicotinic acid adenine dinucleotide phosphate (NAADP) targets in mammalian cells

Yaping Lin-Moshier, Timothy F. Walseth, Dev Churamani, Sean M. Davidson, James T. Slama, Robert Hooper, Eugen Brailoiu, Sandip Patel, Jonathan S. Marchant

Research output: Contribution to journalArticlepeer-review

129 Scopus citations


Nicotinic acid adenine dinucleotide phosphate (NAADP) is an agonist-generated second messenger that releases Ca2+from intracellular acidic Ca2+ stores. Recent evidence has identified the two-pore channels (TPCs) within the endolysosomal system as NAADP-regulated Ca2+ channels that release organellar Ca2+ 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 ([ 32P-5N3]NAADP) that exhibits high affinity binding to NAADP receptors. In several systems that are widely used for studying NAADP-evoked Ca2+ signaling, including sea urchin eggs, human cell lines (HEK293, SKBR3), and mouse pancreas, 5N3-NAADP selectively labeled low molecular weight sites that exhibited the diagnostic pharmacology of NAADP-sensitive Ca2+ 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 knockout 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.

Original languageEnglish (US)
Pages (from-to)2296-2307
Number of pages12
JournalJournal of Biological Chemistry
Issue number4
StatePublished - Jan 20 2012


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