STOML3 FORMS RING-LIKE MEMBRANE CLUSTERS THAT SCAFFOLD MECHANOSENSITIVE ION CHANNELS

Mechanosensation requires specialized ion channels whose activity is tuned by accessory proteins within the lipid bilayer. Stomatin-like protein 3 (STOML3) potentiates mechanosensitive channels including PIEZO1, PIEZO2, and ELKIN1^1–3, yet its membrane topology and the structural basis for channel regulation remain unknown. Here, using tripartite split-GFP complementation and N-glycosylation mapping, we establish that STOML3 adopts a Type II transmembrane topology with its N-terminus cytoplasmic and C-terminus extracellular—distinct from the hairpin-loop topology of related stomatin family members. Multi-scale super-resolution imaging (Airyscan, STED, and MINFLUX nanoscopy) reveals that STOML3 assembles into heterogeneous ring-like structures (0.1–1.6 µm diameter) at cholesterol-enriched membrane domains. Ring formation critically depends on a conserved membrane-proximal proline residue (P44 in human) that mediates cholesterol binding; the P44S mutation abolishes ring architecture without eliminating membrane targeting. We demonstrate that STOML3 directly interacts with and scaffolds PIEZO1, PIEZO2, and ELKIN1 at lipid rafts, constraining PIEZO channels mobility. Disrupting ring formation through P44S mutation, pharmacological inhibition (OB-1), or cholesterol depletion reduces channel interaction by 50–70%, revealing a two-mode interaction model: a ring-independent component mediating ~30–50% of channel association, and a ring-dependent component essential for mechanotransduction. These findings establish STOML3 as a cholesterol-dependent membrane scaffold that organizes mechanosensitive channels through distinct structural and functional interaction modes, identifying ring formation as a selective therapeutic target for mechanical allodynia.