Energetic destabilization of shut states underpins the membrane stretch dependent activation of Piezo1

Piezo1 is a non-selective cation channel activated by mechanical stimuli such as cell membrane stretch. It has been shown that Piezo1 conforms to a curved, upright, bowl-like structure in its non-conductive (“shut”) state. Stretching of the cell membrane is thought to flatten this curved structure of Piezo1 to open its ion-conducting pore. Yet, how stretching of the cell membrane modulates the functional states of Piezo1 to open the channel is not known. Therefore, we collected single-channel Piezo1 recordings at various steady-state pressure-induced membrane stretch to study how the membrane stretch alters functional states of Piezo1. One-dimensional single-channel dwell time histograms reveal that Piezo1 has a minimum of two shut (long-lived and short-lived) and two open (long-lived and short-lived) functional states. Two-dimensional dwell time histograms and Markov modeling show that membrane stretch increases the rate of microscopic shut-to-open transitions, with relatively smaller effects on closure rates. Critically, increasing membrane stretch up to saturation of open probability correlates with a ∼25- and ∼50-fold decrease of mean dwell times for the short-lived and long-lived shut states, respectively. In contrast, the mean dwell times for the short-lived and long-lived open states only increased by ∼3- and ∼2-fold, respectively. Taken together, these results indicate that membrane stretch predominantly acts by energetically destabilizing Piezo1 shut states.