Small molecule agonist TPC2-A1-N increases intracellular Ca2+ independent of Two-Pore Channels

Two-Pore channels (TPCs) constitute a small family of cation channels expressed in endo-lysosomal compartments. TPCs have been characterized as important constituents controlling Ca2+-mediated vesicular membrane fusion and fission, thereby regulating intracellular organelle trafficking. Two activators, NAADP and PI(3,5)P2 induce ion flux through TPCs. The membrane-permeable small molecule activators TPC2-A1-N and TPC2-A1-P have been identified and postulated to mimic their action and to discriminate for a preferential selectivity either for Ca2+ or for Na+. This was observed only for TPC2 and was independent of NAADP-binding proteins. Here, we applied TPC2-A1-N and measured intracellular increase of Ca2+ and Na+ in MEF, HeLa and J774 cells. TPC2-A1-N did not only increase Ca2+ levels in wildtype, but also in all cells with genetically inactivated TPCs. Depletion of Ca2+ from the ER via thapsigargin caused a massive reduction of the TPC2-A1-N induced Ca2+ elevation in all cell lines, indicating that ER plays a key role in this context. Furthermore, our results point to an IP3 receptor independent TPC2-A1-N mediated Ca2+ release. Ca2+ depletion from ER was also observed by using an ER-targeted GCaMP6 construct. TPC2-A1-N also raised Na+ levels in MEF cells deficient for TPC1 and TPC2. In summary, our results suggest that TPC2-A1-N induced Ca2+ and Na+ signals are independent of any TPC and that ER represents the major source of Ca2+.