State-dependent binding of the wedge domain controls inactivation of the mechanosensitive ion channel PIEZO1

The mechanically activated ion channel PIEZO1 transduces membrane tension into intracellular calcium signals and is critical for a wide range of physiological processes. Recent structural and functional studies have established a detailed framework for PIEZO1 activation, but the molecular mechanisms governing its rapid inactivation remain incompletely understood. Here, we examined the contribution of the intracellular wedge domain to PIEZO1 inactivation using site-directed mutagenesis, electrophysiological recordings and MINFLUX nanoscopy. We show that wedge deletion and disruption of specific {pi}-{pi} and cation-{pi} interactions between the wedge 1-helix and the pore module abolishes inactivation without impairing channel activation. Moreover, MINFLUX nanoscopy reveals that the wedge stabilizes a flat inactivated conformation of PIEZO1 and suggests that wedge dissociation is required for recovery from inactivation. Together, our data support a ball-and-chain-like mechanism with the wedge acting as a state-dependent inactivation particle that docks to the pore module to terminate channel activity during sustained mechanical stimulation.