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. Here, MCOLN1 is linked to mucolipidosis type IV.