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EMBO J. 2015 Jul 2;34(13):1743-58. doi: 10.15252/embj.201490009. Epub 2015 Apr 14.

Expression of Ca²⁺-permeable two-pore channels rescues NAADP signalling in TPC-deficient cells.

Author information

1
Department of Pharmacology, University of Oxford, Oxford, UK.
2
Center for Integrated Protein Science CIPS-M and Department of Pharmacy - Center for Drug Research, Ludwig-Maximilians-Universität München, München, Germany.
3
Pharmacology Department, University of Minnesota, Minneapolis, MN, USA.
4
Department of Pharmacology, University of Oxford, Oxford, UK john.parrington@pharm.ox.ac.uk antony.galione@pharm.ox.ac.uk.

Abstract

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.

KEYWORDS:

Ca2+; NAADP; TPC; electrophysiology; endo‐lysosome

PMID:
25872774
PMCID:
PMC4516428
DOI:
10.15252/embj.201490009
[Indexed for MEDLINE]
Free PMC Article

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