A disease mutation reveals a non-swapped pore and rewired gating cycle of the mechanically activated PIEZO1 channel

PIEZO1 is a versatile mechanotranduction channel critical for diverse cellular and physiological processes ^1–3 . Gain-of-function mutations, such as R2482H, which slow channel inactivation, are linked to dehydrated hereditary stomatocytosis (DHS) and iron overload ^4–7 . PIEZO1 forms a gigantic bowl-shaped trimer comprising flattenable peripheral blades and a central pore module with a switchable cap ^8–12 . However, the structural dynamics governing its gating process and pathogenesis remain incompletely understood. Here, we determined distinct conformational states of the DHS-associated PIEZO1-R2482H mutant in both detergents and proteoliposomes. Remarkably, the R2482H mutation in the central pore induces a conformational change that propagates ∼25 nm to the most distal blade, enabling resolution of all 38 transmembrane helices. PIEZO1-R2482H is inherently flatter than PIEZO1, lowering the gating force and energy barrier and thus explaining its heightened mechanosensitivity. While the flattened PIEZO1 has its cap in the up-state adopting an inactivated state, the flattened PIEZO1-R2482H has its cap in the down-state and twisted by ∼75°, drastically switching the pore module from a swapped to a non-swapped organization. Such twisted conformation of PIEZO1-R2482H might hinder the transition to the inactivated state, instead proceeding through a deactivation-transition state characterized by curved blades and a partially twisted cap, thereby accounting for the slowed inactivation and deactivation kinetics. Taken together, we reveal the full transmembrane topology and a non-swapped pore conformation induced by R2482H, and propose the structural dynamic model that delineates the distinct gating cycle of activation, inactivation, and deactivation for PIEZO1 and PIEZO1-R2482H.