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The second messenger NAADP triggers Ca(2+) release from endo-lysosomes. Although two-pore channels (TPCs) have been proposed to be regulated by NAADP, recent studies have challenged this. By generating the first mouse line with demonstrable absence of both Tpcn1 and Tpcn2 expression (Tpcn1/2(-/-)), we show that the loss of endogenous TPCs abolished NAADP-dependent Ca(2+) responses as assessed by single-cell Ca(2+) imaging or patch-clamp of single endo-lysosomes. In contrast, currents stimulated by PI(3,5)P2 were only partially dependent on TPCs. In Tpcn1/2(-/-) cells, NAADP sensitivity was restored by re-expressing wild-type TPCs, but not by mutant versions with impaired Ca(2+)-permeability, nor by TRPML1. Another mouse line formerly reported as TPC-null likely expresses truncated TPCs, but we now show that these truncated proteins still support NAADP-induced Ca(2+) release. High-affinity [(32)P]NAADP binding still occurs in Tpcn1/2(-/-) tissue, suggesting that NAADP regulation is conferred by an accessory protein. Altogether, our data establish TPCs as Ca(2+)-permeable channels indispensable for NAADP signalling.

Original publication

DOI

10.15252/embj.201490009

Type

Journal article

Journal

EMBO J

Publication Date

02/07/2015

Volume

34

Pages

1743 - 1758

Keywords

Ca2+, NAADP, TPC, electrophysiology, endo‐lysosome, Animals, Calcium, Calcium Channels, Calcium Signaling, Cells, Cultured, Evoked Potentials, Gene Expression, Hydrogen-Ion Concentration, Lysosomes, Mice, Mice, Knockout, NADP, Protein Isoforms, Signal Transduction