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

The small molecule Yoda1 has become an indispensable tool for dissecting the role of the mechanically activated ion channel PIEZO1 in physiological and pathological contexts. Previous studies proposed that Yoda1 first binds to a hydrophobic pocket at the interface of the transmembrane helical units 8 and 9 close to the inner leaflet of the membrane to induce conformational flattening and thus channel activation and then transitions to a deeper located binding site with higher affinity in the open conformation. Here, using site-directed mutagenesis, electrophysiology, computational modelling and 3D-MINFLUX nanoscopy, we refine this model by demonstrating that mutation of the previously proposed Yoda1 binding site-I, 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 revised model for Yoda's mode of action that distinguishes discrete allosteric pathways for PIEZO1 activation versus modulation and provides a framework for the design of next-generation use-dependent PIEZO1 modulators.