Voltage-dependent volume regulation controls epithelial cell extrusion and morphology

Epithelial cells work collectively to provide a protective barrier, yet also turn over rapidly by cell death and division. If the number of dying cells does not match those dividing, the barrier would vanish, or tumors can form. Mechanical forces and the stretch-activated ion channel (SAC) Piezo1 link both processes; stretch promotes cell division and crowding triggers cell death by initiating live cell extrusion1,2. However, it was not clear how particular cells within a crowded region are selected for extrusion. Here, we show that individual cells transiently shrink via water loss before they extrude. Artificially inducing cell shrinkage by increasing extracellular osmolarity is sufficient to induce cell extrusion. Pre-extrusion cell shrinkage requires the voltage-gated potassium channels Kv1.1 and Kv1.2 and the chloride channel SWELL1, upstream of Piezo1. Activation of these voltage-gated channels requires the mechano-sensitive Epithelial Sodium Channel, ENaC, acting as the earliest crowd-sensing step. Imaging with a voltage dye indicated that epithelial cells lose membrane potential as they become crowded and smaller, yet those selected for extrusion are markedly more depolarized than their neighbours. Loss of any of these channels in crowded conditions causes epithelial buckling, highlighting an important role for voltage and water regulation in controlling epithelial shape as well as extrusion. Thus, ENaC causes cells with similar membrane potentials to slowly shrink with compression but those with reduced membrane potentials to be eliminated by extrusion, suggesting a chief driver of cell death stems from insufficient energy to maintain cell membrane potential.