Differential ion selectivity and disease-associated dysfunction of TRPML channels revealed by patient and engineered...

The endolysosomal transient receptor potential mucolipin (TRPML) channels play key roles in regulating lysosomal trafficking, signaling, and function. While mutations in TRPML1 cause mucolipidosis type IV (MLIV), the functional consequences of many disease-associated mutations remain unclear. In this study, we used live-cell confocal imaging in HeLa cells to comprehensively characterize the subcellular localization and cation (Ca²⁺ and Zn²⁺) permeability of TRPML1-3, 10 TRPML1 MLIV patient-derived mutants, and engineered pore mutants. We showed that TRPML1 and TRPML3 permeate both Ca²⁺ and Zn²⁺, whereas TRPML2 conducts only Ca²⁺. Subcellular localization analyses revealed that TRPML1 and TRPML2 localize predominantly to lysosomes, whereas TRPML3 is preferentially localized to the endoplasmic reticulum. Among the 10 patient-derived TRPML1 mutants, nine exhibited severely impaired agonist-mediated Ca²⁺ and Zn²⁺ permeability, indicating severe functional loss. In contrast, the F408Δ mutant, associated with a milder phenotype, retained partial ion permeability and was the only mutant capable of constitutive Ca²⁺ permeation without agonist stimulation. Interestingly, we found that nonfunctional, lysosome-localized TRPML1 mutants are associated with more severe disease phenotypes than those retained in the endoplasmic reticulum, suggesting that lysosomal localization of nonfunctional TRPML1 may have dominant-negative or toxic effects. Finally, through structure-guided mutagenesis, we generated a metal-selective TRPML1 mutant, I468V, that is permeable to Ca²⁺ but not to Zn²⁺, providing a new tool for dissecting the distinct physiological roles of Zn²⁺ and Ca²⁺ in TRPML1-mediated processes. Together, these findings provide new insights into how TRPML1 mutations disrupt subcellular localization, ion permeability, and selectivity, which contribute to the variable clinical spectrum of MLIV.