Segregated cation flux by TPC2 biases Ca 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)P. Whereas NAADP rendered the channel Ca-permeable and PI(3,5)P rendered the channel Na-selective, a combination of the two increased Ca but not Na flux. Mechanistically, this was due to an increase in Ca permeability independent of changes in ion selectivity. Functionally, we show that cell permeable NAADP and PI(3,5)P mimetics synergistically activate native TPC2 channels in live cells, globalizing cytosolic Ca 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 Ca signaling.