TRPML2 channel modulation by PI(3,5)P₂ and small-molecule agonists controls endosomal vesicle dynamics

TRPML2 is an endolysosomal calcium-permeable channel gated by phosphatidylinositol 3,5-bisphosphate (PI(3,5)P₂). However, its subcellular localization and functional contribution to compartment-specific vesicle trafficking remain incompletely defined. In this study, we identify Rab4-positive recycling endosomes as a key site of TRPML2 activity and regulation. We further examined a PI(3,5)P₂-insensitive TRPML2 mutant (R310A), which exhibited reduced channel activity, accumulated perinuclear vesicles, and impaired Rab4 + endosomal motility. To restore channel function, we employed two selective small-molecule TRPML2 agonists, ML2-SA1 and ML2-SA2. Endolysosomal patch-clamp recordings confirmed that both compounds activate wild-type TRPML2 and effectively restore channel activity in the R310A mutant. Functional imaging further demonstrated that ML2-SA2 treatment rescues vesicle redistribution, reduces Golgi accumulation, and promotes peripheral vesicle dynamics. Notably, these effects were observed even in the absence of functional PI(3,5)P₂ gating, indicating that small-molecule agonists can bypass endogenous lipid regulation to restore TRPML2 activity. Our findings identify TRPML2 as a druggable ion channel whose activity is essential for maintaining Rab4-dependent vesicle trafficking. This study establishes a mechanistic link between phosphoinositide sensitivity, TRPML2 activation, and endosomal motility, and highlights a potential therapeutic strategy for correcting trafficking defects caused by impaired lipid signaling in immune cells or other pathophysiological contexts involving endosomal stress.