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

The second messenger NAADP triggers Ca²⁺ 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 Ca²⁺ responses as assessed by single-cell Ca²⁺ 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 Ca²⁺-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 Ca²⁺ release. High-affinity [³²P]NAADP binding still occurs in Tpcn1/2^-/- tissue, suggesting that NAADP regulation is conferred by an accessory protein. Altogether, our data establish TPCs as Ca²⁺-permeable channels indispensable for NAADP signalling. Synopsis By presenting a new double-knockout mouse model this study demonstrates that two-pore channels (TPCs) are responsible for NAADP-dependent Ca²⁺ release from endo-lysosomes. Endogenous two-pore channels (TPCs) are essential for NAADP-stimulated Ca²⁺ currents and Ca²⁺ release. Re-expression of TPCs in validated null-background cells restores NAADP sensitivity. Truncated TPC1 and TPC2 proteins - potentially expressed in other reported TPC knockouts - are still functional. TPCs are not required for high-affinity NAADP-binding. PI(3,5)P₂ is a promiscuous modulator of endo-lysosomal channels. By presenting a new double-knockout mouse model this study demonstrates that two-pore channels (TPCs) are responsible for NAADP-dependent Ca²⁺ release from endo-lysosomes.