TRPML3/BK complex promotes autophagy and bacterial clearance by providing a positive feedback regulation of mTOR via PI3P.

TRPML3 is a Ca2+/Na+ release channel residing in both phagophores and endolysosomal membranes. It is activated by PI3P and PI3,5P2. Its activity can be enhanced by high luminal pH and by replacing luminal Na+ with K+. Here, we report that big-conductance Ca2+-activated potassium (BK) channels form a positive feedback loop with TRPML3. Ca2+ release via TRPML3 activates BK, which in turn facilitates TRPML3-mediated Ca2+ release, potentially through removing luminal Na+ inhibition. We further show that TRPML3/BK and mammalian target of rapamycin (mTOR) form another positive feedback loop to facilitate autophagy induction in response to nutrient starvation, i.e., mTOR inhibition upon nutrient starvation activates TRPML3/BK, and this further reduces mTOR activity, thereby increasing autophagy induction. Mechanistically, the feedback regulation between TRPML3/BK and mTOR is mediated by PI3P, an endogenous TRPML3 activator that is enriched in phagophores and is up-regulated by mTOR reduction. Importantly, bacterial infection activates TRPML3 in a BK-dependent manner, and both TRPML3 and BK are required for mTOR suppression and autophagy induction responding to bacterial infection. Suppressing either TRPML3 or BK helps bacteria survival whereas increasing either TRPML3 or BK favors bacterial clearance. Considering that TRPML3/BK is inhibited by low luminal pH but activated by high luminal pH and PI3P in phagophores, we suggest that TRPML3/BK and mTOR form a positive feedback loop via PI3P to ensure efficient autophagy induction in response to nutrient deprivation and bacterial infection. Our study reveals a role of TRPML3-BK coupling in controlling cellular homeostasis and intracellular bacterial clearance via regulating mTOR signaling.

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.