Segregated cation flux by TPC2 biases Ca²⁺ signaling through lysosomes

Two-pore channels are endo-lysosomal cation channels with malleable selectivity filters that drive endocytic ion flux and membrane traffic. Here we show that TPC2 can differentially regulate its cation permeability when co-activated by its endogenous ligands, NAADP and PI(3,5)P₂. Whereas NAADP rendered the channel Ca²⁺-permeable and PI(3,5)P₂ rendered the channel Na⁺-selective, a combination of the two increased Ca²⁺ but not Na⁺ flux. Mechanistically, this was due to an increase in Ca²⁺ permeability independent of changes in ion selectivity. Functionally, we show that cell permeable NAADP and PI(3,5)P₂ mimetics synergistically activate native TPC2 channels in live cells, globalizing cytosolic Ca²⁺ signals and regulating lysosomal pH and motility. Our data reveal that flux of different ions through the same pore can be independently controlled and identify TPC2 as a likely coincidence detector that optimizes lysosomal Ca²⁺ signaling.