In contrast to the plasma membrane, the vacuole membrane has not yet been associated with electrical excitation of plants. Here, we show that mesophyll vacuoles from Arabidopsis sense and control the membrane potential essentially via the K⁺-permeable TPC1 and TPK channels. Electrical stimuli elicit transient depolarization of the vacuole membrane that can last for seconds. Electrical excitability is suppressed by increased vacuolar Ca²⁺ levels. In comparison to wild type, vacuoles from the fou2 mutant, harboring TPC1 channels insensitive to luminal Ca²⁺, can be excited fully by even weak electrical stimuli. The TPC1-loss-of-function mutant tpc1-2 does not respond to electrical stimulation at all, and the loss of TPK1/TPK3-mediated K⁺ transport affects the duration of TPC1-dependent membrane depolarization. In combination with mathematical modeling, these results show that the vacuolar K⁺-conducting TPC1 and TPK1/TPK3 channels act in concert to provide for Ca²⁺- and voltage-induced electrical excitability to the central organelle of plant cells.

Lysosomes are cell organelles that degrade macromolecules to recycle their components. If lysosomal degradative function is impaired, e.g., due to mutations in lysosomal enzymes or membrane proteins, lysosomal storage diseases (LSDs) can develop. LSDs manifest often with neurodegenerative symptoms, typically starting in early childhood, and going along with a strongly reduced life expectancy and quality of life. We show here that small molecule activation of the Ca²⁺‐permeable endolysosomal two‐pore channel 2 (TPC2) results in an amelioration of cellular phenotypes associated with LSDs such as cholesterol or lipofuscin accumulation, or the formation of abnormal vacuoles seen by electron microscopy. Rescue effects by TPC2 activation, which promotes lysosomal exocytosis and autophagy, were assessed in mucolipidosis type IV (MLIV), Niemann–Pick type C1, and Batten disease patient fibroblasts, and in neurons derived from newly generated isogenic human iPSC models for MLIV and Batten disease. For in vivo proof of concept, we tested TPC2 activation in the MLIV mouse model. In sum, our data suggest that TPC2 is a promising target for the treatment of different types of LSDs, both in vitro and in‐vivo.

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.

Transient receptor potential (TRP) or transient receptor potential channels are a highly diverse family of mostly non-selective cation channels. In the mammalian genome, 28 members can be identified, most of them being expressed predominantly in the plasma membrane with the exception of the mucolipins or TRPMLs which are expressed in the endo-lysosomal system. In mammalian organisms, TRPMLs have been associated with a number of critical endo-lysosomal functions such as autophagy, endo-lysosomal fusion/fission and trafficking, lysosomal exocytosis, pH regulation, or lysosomal motility and positioning. The related non-selective two-pore cation channels (TPCs), likewise expressed in endosomes and lysosomes, have also been found to be associated with endo-lysosomal trafficking, autophagy, pH regulation, or lysosomal exocytosis, raising the question why these two channel families have evolved independently. We followed TRP/TRPML channels and TPCs through evolution and describe here in which species TRP/TRPMLs and/or TPCs are found, which functions they have in different species, and how this compares to the functions of mammalian orthologs.

Lysosomal storage diseases (LSDs) resulting from inherited gene mutations constitute a family of disorders that disturb lysosomal degradative function leading to abnormal storage of macromolecular substrates. In most LSDs, central nervous system (CNS) involvement is common and leads to the progressive appearance of neurodegeneration and early death. A growing amount of evidence suggests that ion channels in the endolysosomal system play a crucial role in the pathology of neurodegenerative LSDs. One of the main basic mechanisms through which the endolysosomal ion channels regulate the function of the endolysosomal system is Ca²⁺ release, which is thought to be essential for intracellular compartment fusion, fission, trafficking and lysosomal exocytosis. The intracellular TRPML (transient receptor potential mucolipin) and TPC (two-pore channel) ion channel families constitute the main essential Ca²⁺-permeable channels expressed on endolysosomal membranes, and they are considered potential drug targets for the prevention and treatment of LSDs. Although TRPML1 activation has shown rescue effects on LSD phenotypes, its activity is pH dependent, and it is blocked by sphingomyelin accumulation, which is characteristic of some LSDs. In contrast, TPC2 activation is pH-independent and not blocked by sphingomyelin, potentially representing an advantage over TRPML1. Here, we discuss the rescue of cellular phenotypes associated with LSDs such as cholesterol and lactosylceramide (LacCer) accumulation or ultrastructural changes seen by electron microscopy, mediated by the small molecule agonist of TPC2, TPC2-A1-P, which promotes lysosomal exocytosis and autophagy. In summary, new data suggest that TPC2 is a promising target for the treatment of different types of LSDs such as MLIV, NPC1, and Batten disease, both in vitro and in vivo.