Yoda molecules agonize PIEZO2

PIEZO proteins (PIEZO1 and PIEZO2) are essential mechanosensitive channels. PIEZO1 is thought to be selectively activated by Yoda molecules (Yoda1 and Yoda2). Although a structural framework for PIEZO1 activation by Yoda1 exists, a molecular mechanism underlying this selective activation is lacking. Here, using electrophysiology and calcium imaging, we show that Yoda1 increases PIEZO2 open probability and stretch sensitivity as efficaciously as PIEZO1 but elicits weaker PIEZO2-dependent calcium entry, rationalizing why its effect on PIEZO2 has been overlooked. Both Yoda1 and its more potent Yoda2 analog slow down inactivation of PIEZO2 currents with potency similar to PIEZO1 but with lower efficacy. Using mutagenesis and molecular dynamics simulations, we further show that Yoda2’s benzoic acid group forms a transient salt bridge with a conserved arginine in the Yoda binding site, providing a molecular basis for Yoda2’s increased potency. Our study cautions a reevaluation of studies using these molecules to untangle biological functions mediated by PIEZO channels.