3D-MINFLUX nanoscopy reveals distinct allosteric mechanisms for activation and modulation of PIEZO1 by Yoda1

PIEZO1 underpins numerous physiological processes by which cells detect and respond to mechanical stimuli. The small molecule Yoda1 has become an indispensable tool that is frequently used for dissecting the role of PIEZO1 in physiological and pathological contexts, yet its mode of action remains incompletely understood. Here, using site-directed mutagenesis, electrophysiology, computational modelling and 3D-MINFLUX nanoscopy, we demonstrate that mutation of the previously proposed Yoda1 binding site, solely abolishes Yoda1-induced activation and channel flattening but preserves modulation of mechanically-evoked PIEZO1 currents, whereas mutation of F1715, which lines a transient binding cavity accessible only in the flattened PIEZO1 conformation, eliminated modulation without affecting Yoda-induced calcium entry. Thus, our data support a two-site induced-fit-like mechanism where Yoda first engages binding site-1 to promote blade flattening and pore opening and then transitions to a deeper binding site to modulate the mechanical activation threshold and inactivation kinetics. This revised model distinguishes discrete allosteric pathways for PIEZO1 activation versus modulation and provides a framework for the design of next-generation use-dependent PIEZO1 modulators.