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, nicotinic acid adenine dinucleotide phosphate and phosphatidylinositol-3,5-bisphosphate, 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 nicotinic acid adenine dinucleotide phosphate-binding proteins. Here, we applied TPC2-A1-N and measured intracellular increase of Ca²⁺ and Na⁺ in mouse embryonic fibroblast, HeLa, and J774 cells. TPC2-A1-N did not only increase Ca²⁺ levels in WT but also in all cells with genetically inactivated TPCs. Depletion of Ca²⁺ from the endoplasmic reticulum (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 inositol triphosphate 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 mouse embryonic fibroblast 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⁺.