Yoda molecules agonize PIEZO2

Mammalian PIEZO proteins (PIEZO1 and PIEZO2) act as essential mechanosensitive channels. PIEZO1 is thought to be selectively activated by Yoda molecules (Yoda1 and Yoda2). Although a molecular mechanism for PIEZO1 activation by Yoda1 exists, the origin of Yoda1 selective agonism remains obscure. Here, using electrophysiology and calcium imaging in transfected cells, we show that Yoda1 agonizes PIEZO2 but with a distinct efficacy profile compared to PIEZO1. Yoda1 increases single channel open probability and macroscopic stretch sensitivity of PIEZO2 currents as efficaciously as reported for PIEZO1, but it elicits weak PIEZO2-dependent calcium influx in absence of external stimulus, an effect detectable only under non-physiological conditions. Yoda1 slows down inactivation of indentation-evoked PIEZO2 currents, but to a lower extent compared to PIEZO1. The Yoda1 analog Dooku1, which does not modulate PIEZO1, slows down PIEZO2 inactivation similarly to Yoda1. Two PIEZO1 agonists unrelated to Yoda1 but thought to populate the same binding site in PIEZO1 have no effect on PIEZO2 inactivation, suggesting that their PIEZO1 selectivity is better than Yoda1. PIEZO2 inactivation is also modulated by Yoda1 potent analog Yoda2. Using mutagenesis and molecular dynamics simulations, we show that Yoda2 benzoic acid group forms a transient salt bridge with a conserved arginine in the Yoda binding site (R1724/R2029 in mouse PIEZO1/PIEZO2), explaining the higher potency of this analog for both channels. Together, our study re-evaluates the PIEZO1 selectivity of Yoda molecules and highlights a complex modulatory landscape toward distinct functional properties of PIEZO channels.