Removal of the mechanoprotective influence of the cytoskeleton reveals PIEZO1 is gated by bilayer tension

TLDR

Mechanosensitive ion channels transduce mechanical stimuli into ion flux, yet their gating mechanisms are difficult to study because forces are shared among the membrane, cytoskeleton, and extracellular matrix. The study tests whether PIEZO1 is activated by force transmitted directly through the bilayer. HEK293 membrane blebs largely devoid of cytoskeleton were generated, and bilayer tension was calibrated using bacterial MscL to confirm that bleb activation matches that in reconstituted bilayers. PIEZO1–GFP fusion experiments revealed that PIEZO1 gates at lower pressures in bleb membranes, demonstrating that it senses bilayer tension directly and that the cortical cytoskeleton provides mechanoprotection.

Abstract

Abstract Mechanosensitive ion channels are force-transducing enzymes that couple mechanical stimuli to ion flux. Understanding the gating mechanism of mechanosensitive channels is challenging because the stimulus seen by the channel reflects forces shared between the membrane, cytoskeleton and extracellular matrix. Here we examine whether the mechanosensitive channel PIEZO1 is activated by force-transmission through the bilayer. To achieve this, we generate HEK293 cell membrane blebs largely free of cytoskeleton. Using the bacterial channel MscL, we calibrate the bilayer tension demonstrating that activation of MscL in blebs is identical to that in reconstituted bilayers. Utilizing a novel PIEZO1–GFP fusion, we then show PIEZO1 is activated by bilayer tension in bleb membranes, gating at lower pressures indicative of removal of the cortical cytoskeleton and the mechanoprotection it provides. Thus, PIEZO1 channels must sense force directly transmitted through the bilayer.