Membrane fusion and fission events in intracellular trafficking are controlled by both intraluminal Ca²⁺ release and phosphoinositide (PIP) signalling. However, the molecular identities of the Ca²⁺ release channels and the target proteins of PIPs are elusive. In this paper, by direct patch-clamping of the endolysosomal membrane, we report that PI(3,5)P₂, an endolysosome-specific PIP, binds and activates endolysosome-localized mucolipin transient receptor potential (TRPML) channels with specificity and potency. Both PI(3,5)P₂-deficient cells and cells that lack TRPML1 exhibited enlarged endolysosomes/vacuoles and trafficking defects in the late endocytic pathway. We find that the enlarged vacuole phenotype observed in PI(3,5)P₂-deficient mouse fibroblasts is suppressed by overexpression of TRPML1. Notably, this PI(3,5)P₂-dependent regulation of TRPML1 is evolutionarily conserved. In budding yeast, hyperosmotic stress induces Ca²⁺ release from the vacuole. In this study, we show that this release requires both PI(3,5)P₂ production and a yeast functional TRPML homologue. We propose that TRPMLs regulate membrane trafficking by transducing information regarding PI(3,5)P₂ levels into changes in juxtaorganellar Ca²⁺, thereby triggering membrane fusion/fission events.

Lysosomal lipid accumulation, defects in membrane trafficking and altered Ca²⁺ homoeostasis are common features in many lysosomal storage diseases. Mucolipin transient receptor potential channel 1 (TRPML1) is the principle Ca²⁺ channel in the lysosome. Here we show that TRPML1-mediated lysosomal Ca²⁺ release, measured using a genetically encoded Ca²⁺ indicator (GCaMP3) attached directly to TRPML1 and elicited by a potent membrane-permeable synthetic agonist, is dramatically reduced in Niemann–Pick (NP) disease cells. Sphingomyelins (SMs) are plasma membrane lipids that undergo sphingomyelinase (SMase)-mediated hydrolysis in the lysosomes of normal cells, but accumulate distinctively in lysosomes of NP cells. Patch-clamp analyses revealed that TRPML1 channel activity is inhibited by SMs, but potentiated by SMases. In NP-type C cells, increasing TRPML1's expression or activity was sufficient to correct the trafficking defects and reduce lysosome storage and cholesterol accumulation. We propose that abnormal accumulation of luminal lipids causes secondary lysosome storage by blocking TRPML1- and Ca²⁺-dependent lysosomal trafficking.