Data from: Agonist-specific voltage-dependent gating of lysosomal Two-Pore Na + Channels

Mammalian Two-Pore-Channels (TPC1, 2; TPCN1, TPCN2) are ubiquitously-expressed cation channels in the endosomes and lysosomes that regulate luminal pH homeostasis, membrane trafficking, and Ebola viral infection. Lysosomal patch-clamp studies and high-resolution structural determinations have demonstrated that TPCs are Na+-selective channels activated by lysosome-localized PI(3,5)P2. Whereas the channel activity of TPC1 is strongly dependent on membrane voltage, TPC2 lacks such voltage dependence despite the presence of several positively-charged amino acid (AA) residues in the presumed "S4 voltage-sensing" domains. By performing a Ca2+-imaging-based high-throughput screening followed by lysosomal electrophysiology, here we identified a class of tri-cyclic anti-depressants (TCAs) as small-molecule agonists of TPC channels. TCAs activate both TPC1 and TPC2 in a voltage-dependent manner, so we refer to them as Lysosomal Na+ channel Voltage-dependent Activators (LyNa-VAs). We also identified another compound which, like PI(3,5)P2, activates TPC2 independent of voltage, suggesting the existence of agonist-specific gating mechanisms. Our identification of small-molecule TPC agonists should facilitate the studies of the cell biological roles of TPCs, and can also readily explain the reported effects of TCAs in the modulation of autophagy and lysosomal function.