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Expression of Ca²⁺-permeable two-pore channels rescues NAADP signalling in TPC-deficient cells
Margarida Ruas1, Lianne C Davis1, Cheng-Chang Chen2
1Department of Pharmacology, University of Oxford, Oxford, 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.
Insights
Two-pore channels (TPCs) are essential for NAADP-mediated calcium release from endo-lysosomes. This study confirms TPCs
Area of Science:
- Cell Biology
- Molecular Biology
- Physiology
Background:
- Nicotinic acid adenine dinucleotide phosphate (NAADP) is a key second messenger that mobilizes intracellular calcium (Ca2+) stores.
- Endo-lysosomes are critical organelles involved in cellular signaling, and their Ca2+ dynamics are tightly regulated.
- Two-pore channels (TPCs) have been implicated as NAADP receptors, but their precise role remains debated.
Purpose of the Study:
- To definitively establish the role of endogenous two-pore channels (TPCs) in NAADP-mediated Ca2+ signaling.
- To investigate the functional consequences of TPC absence on endo-lysosomal Ca2+ release.
- To identify potential accessory proteins involved in NAADP regulation.
Main Methods:
- Generation and characterization of a novel double knockout mouse line lacking both Tpcn1 and Tpcn2 expression (Tpcn1/2(-/-)).
- Single-cell Ca2+ imaging and patch-clamp electrophysiology to assess Ca2+ fluxes.
- Re-expression of wild-type and mutant TPCs in knockout cells.
- Analysis of PI(3,5)P2-stimulated currents.
- Radioligand binding assays using [(32)P]NAADP.
Main Results:
- Loss of endogenous TPCs in Tpcn1/2(-/-) mice completely abolished NAADP-dependent Ca2+ release from endo-lysosomes.
- NAADP-induced Ca2+ responses were restored by re-expressing wild-type TPCs, but not by Ca2+-impermeable mutants or TRPML1.
- PI(3,5)P2-stimulated currents showed partial dependence on TPCs.
- High-affinity NAADP binding persisted in TPC-null tissues, suggesting the involvement of an accessory protein.
Conclusions:
- TPCs are indispensable Ca2+-permeable channels for NAADP signaling.
- TPCs are the primary mediators of NAADP-evoked Ca2+ release from endo-lysosomes.
- An accessory protein likely confers NAADP sensitivity to TPCs.
