Probing the Mechanosensitivity of Piezo1 Channels

Abstract

Piezo channels are a new family of cation-selective mechanosensitive ion channels, which have been shown to be integral in numerous physiological processes. Central to these physiological roles is the ability to sense mechanical force. Whether this force is transmitted directly from the lipid bilayer or from tethering to the cytoskeleton and/or ECM is unknown. The ‘gold standard’ for testing the ‘inherent’ mechanosensitivity of ion channels has become purification and subsequent reconstitution of mechanosensitive channel proteins into liposomes of known lipid composition. This is experimentally difficult and time consuming and is a process that has to be determined empirically for individual channel proteins. An underused paradigm for the study of channel mechanosensitivity is the production of cytoskeleton deficient membrane ‘blebs’ [1]. In order to study the inherent mechanosensitivity of Piezo1 channels using this paradigm we used transiently transfected HEK293 cells. Herein we show that; a) membrane blebs can be formed by the addition of both Hypo- and Hyper-osmolar solutions of sodium gluconate in HEK293 cells, b) this treatment induces significant cell death after 2 hours as determined by trypan blue exclusion assays, c) these resulting blebs are deficient in a major cytoskeletal component (actin) and d) Piezo1 channels can be activated in this environment and have a lower pressure threshold of activation. This has important implications with respect to Piezo1 channels being gated according to the force from lipids concept [2]. In addition we also assess the affect of mutating a number of aromatic residues in the most highly conserved region of the Piezo sequence on the mechanosensitivity of the channel.