Piezo proteins are transmembrane ion channels which transduce many forms of mechanical stimuli into electrochemical signals. Their pore, formed by the assembly of three identical subunits, opens by an unknown mechanism. Here, to probe this mechanism, we investigate the interaction of Piezo1 with the small molecule agonist Yoda1. By engineering chimeras between mouse Piezo1 and its Yoda1-insensitive paralog Piezo2, we first identify a minimal protein region required for Yoda1 sensitivity. We next study the effect of Yoda1 on heterotrimeric Piezo1 channels harboring wild type subunits and Yoda1-insensitive mutant subunits. Using calcium imaging and patch-clamp electrophysiology, we show that hybrid channels harboring as few as one Yoda1-sensitive subunit exhibit Yoda1 sensitivity undistinguishable from homotrimeric wild type channels. Our results show that the Piezo1 pore remains fully open if only one subunit remains activated. This study sheds light on the gating and pharmacological mechanisms of a member of the Piezo channel family.