NAADP-evoked Ca signals through two-pore channel-1 require arginine residues in the first S4-S5 linker.

Two-pore channels (TPCs) are two-domain members of the voltage-gated ion channel superfamily that localize to acidic organelles. Their mechanism of activation (ligands such as NAADP/PI(3,5)P versus voltage) and ion selectivity (Ca versus Na) is debated. Here we report that a cluster of arginine residues in the first domain required for selective voltage-gating of TPC1 map not to the voltage-sensing fourth transmembrane region (S4) but to a cytosolic downstream region (S4-S5 linker). These residues are conserved between TPC isoforms suggesting a generic role in TPC activation. Accordingly, mutation of residues in TPC1 but not the analogous region in the second domain prevents Ca release by NAADP in intact cells. Our data affirm the role of TPCs in NAADP-mediated Ca signalling and unite differing models of channel activation through identification of common domain-specific residues.