Regulation of TRPML1 by Lipids in Lysosomes.

Abstract

TRPML1 is an inwardly-rectifying Ca2+-permeable channel in late endosomes and lysosomes (LELs). Loss-of-function mutations on the human TRPML1 gene cause a devastating pediatric neurodegenerative disease called type IV Mucolipidosis (ML4). Although it is well established that TRPML1 is involved in multiple late endocytic membrane trafficking processes, the regulatory mechanism for TRPML1 remains elusive. By directly patch-clamping endolysosomal membranes, we found that PI(3,5)P2, a low abundance endolysosome-specific phosphoinositide, potently and specifically activates TRPML1 in both heterologous and endogenous systems (Chapter 2). Lipid-protein binding assays showed that PI(3,5)P2 binds directly to the poly-basic region of the N terminus of TRPML1. Overexpression of TRPML1 rescued the enlarged vacuole phenotype in PI(3,5)P2-deficient cells, suggesting that TRPML1 is a downstream effector of PI(3,5)P2. Notably, this PI(3,5)P2-dependent regulation of TRPML1 is evolutionarily conserved. In budding yeast, the activity of a yeast functional TRPML homologue is also PI(3,5)P2-dependent. These results indicate that lysosomal PI(3,5)P2 is a physiological regulator of TRPML1, providing a previously unknown link between these two important regulators of intracellular membrane trafficking. In Chapter 3, a membrane-permeable small-molecule synthetic agonist for TRPMLs, Mucolipin-Synthetic Agonist 1 (ML-SA1), was identified. Electrophysiological results showed that ML-SA1 potently and specifically activates recombinant TRPMLs, as well as endogenous TRPML-like currents in many cell types. In addition, ML-SA1 evoked TRPML1-dependent Ca2+ release from endolysosomes in intact cells. ML-SA1 can therefore serve as a valuable tool for studying intracellular functions of TRPMLs. By taking advantage of ML-SA1, the activity of TRPML1 was examined under a pathological context (Chapter 4). TRPML1-mediated lysosomal Ca2+ release, measured using a genetically-encoded Ca2+ indicator (GCaMP3) attached directly to TRPML1, was dramatically reduced in Niemann-Pick (NP) disease cells. Patch-clamp analyses revealed that TRPML1 channel activity was inhibited by sphingomyelin, but potentiated by spingomyelinase. Importantly, increasing the expression/activity of TRPML1 was able to alleviate the lipid accumulation and trafficking defects in NPC cells. Our findings suggest that compromised channel activity of TRPML1 is the pathogenic cause for secondary lysosomal storage seen in many lysosomal storage disorders (LSDs). Thus manipulating TRPML1 channel activity by chemical agonists may provide therapeutic approaches not only for ML4, but also for other LSDs.