mTOR Regulates Lysosomal ATP-Sensitive Two-Pore Na+ Channels to Adapt to Metabolic State

Abstract

SUMMARY Survival in the wild requires organismal adaptations to the availability of nutrients. Endosomes and lysosomes are key intracellular organelles that couple nutrition and metabolic status to cellular responses, but how they detect cytosolic ATP levels is not well understood. Here we identify an endolysosomal ATP-sensitive Na+ channel (lysoNaATP). The channel is a complex formed by Two-Pore Channels (TPC1 and TPC2), ion channels previously thought to be gated by nicotinic acid adenine dinucleotide phosphate (NAADP), and the mammalian target of rapamycin (mTOR). The channel complex detects nutrient status, becomes constitutively open upon nutrient removal and mTOR translocation off the lysosomal membrane, and controls the lysosome's membrane potential, pH stability, and the amino acid homeostasis. Mutant mice lacking lysoNaATP have much reduced exercise endurance after fasting. Thus, TPCs are a new ion channel family that couple the cell's metabolic state to endolysosomal function and are crucial for physical endurance during food restriction.

Up-regulation of lysosomal TRPML1 channels is essential for lysosomal adaptation to nutrient starvation

“…Two-pore (TPC) Na + -selective channels have recently been proposed to be components of nutrient-sensing machinery in the cell (34). Both ML1 and TPC channels are activated by PI(3,5)P 2 (26,27).…”

High susceptibility to fatty liver disease in two-pore channel 2-deficient mice

“…In this scenario, cations other than Ca 2 þ flowing through TPC2 would act on a downstream channel, which in turn provides Ca 2 þ . Na þ and K þ have also been shown to permeate through TPC2 channels 17,20 . Given the proposed close structural assembly of TPCs and the fusion machinery ( Supplementary Fig.…”

“…In order to allow clearance of early endocytic compartments and ensure specific localization of the dye in the late endocytic pathway, cells were chased in DMEM at 37°C for 1 h in the presence of bovine serum albumin followed by 1-h incubation in serum-free medium before imaging for basal pH determination. To assess lysosomal pH under starvation conditions, the chase procedure was replaced by either 2-h incubation without amino acids (EBSS) or 2-h incubation without both, amino acids and glucose (110 mM NaCl, 45 mM NaHCO 3 , 5 mM KCl, 2 mM CaCl 2 , 1 mM MgCl 2 , pH 7.4) 20 . As a control for increased lysosomal pH, 200 nM Baf.…”

“…There is evidence that TPC1 is mainly present in the proximal endosomal system, and TPC2 is predominantly expressed on late endosomes and lysosomes 16,18,19 . The activation mechanism of TPCs is complex and has been suggested to involve the second messenger nicotinic acid adenine dinucleotide phosphate (NAADP) and the endolysosomal membrane lipidphosphatidylinositol (3,5)bisphosphate (PI(3,5)P 2 ) 17,18,[20][21][22][23][24] . Independent of what the nature of the endogenous ligand of these channels might be, there is substantial evidence that on activation TPCs mediate the release of Ca 2 þ from lysosomal stores.…”

A small molecule restores function to TRPML1 mutant isoforms responsible for mucolipidosis type IV

“…Whole-lysosome planar patch-clamp recordings and preparation of lysosomes (HEK293, Human Fibroblast) were performed as described previously 37,38 . In brief, late endosomes and lysosomes were enlarged by treating the cells with 1 mM vacuolin-1 overnight 12,37,38,49 . The following day, cells were homogenized in homogenization buffer (0.25 M sucrose, 10 mM Tris-Cl, pH 7.4) on ice using a potter homogenizer to obtain whole-cell lysates.…”