O-linked glycan-dependent gating of TPC2 controls lysosomal excitability and organelle remodeling

Two-pore channel 2 (TPC2) is a lysosomal cation channel involved in calcium and sodium signaling and membrane remodeling. Its dysfunction has been linked to diseases including viral infections, neurodegenerative disorders, and cancer. Here, we identify mucin-type O-linked glycosylation at two conserved luminal residues of TPC2, Ser612 and Ser613, as a structural gating brake. Combining structure-guided mutagenesis, lysosomal patch-clamp recordings, calcium nanodomain imaging, molecular dynamics simulations, and small-molecule modulation, we show that genetic, enzymatic, or pharmacological glycan removal enhances basal and ligand-evoked TPC2 activity. This hyperactivation induces lysosomal tubulation, increases vesicle mobility, and promotes TPC2 clustering. In cancer-related models, glycan-deficient TPC2 also increases cell migration, which can be reversed by inhibitors such as tetrandrine and SC-3. These findings establish luminal glycosylation as a key post-translational regulator of TPC2 gating and reveal a mechanism by which ion channel activity controls lysosomal architecture and disease-relevant cell behavior. Lysosomal ion channels regulate diverse cellular processes, yet their modulation remains poorly understood. Here, authors show that mucin-type O-linked glycosylation restrains TPC2 gating, revealing a mechanism linking glycan modification to lysosomal function.