Investigating the gating mechanism of the large conductance mechanosensitive channel by SDSL and EPR spectroscopy

Abstract

Mechanosensation is the ability of cells to sense and respond to mechanical stimuli deriving from their environment. Despite being a fundamental cellular response though, the molecular basis of mechanotransduction is still largely unknown. The mechanosensitive (MS) channel of large conductance, MscL, is expressed by all prokaryotes, it is the MS channel with the highest pressure activation threshold and acts as a last resort safety valve. It responds to the lateral tension of the membrane and, by opening its pore of ≈35Å, it rapidly allows solutes to exit the cell in order to rescue it from lysis during severe hypoosmotic shock. Despite its importance and abundance, the gating and activation mechanism of MscL has not been elucidated. Thus, in the main project presented, MscL’s gating mechanism is investigated as a means to gain insight on the mode of mechanosensation itself. Activation of the channel through site-directed spin labelling (SDSL) reveals that the nano-pockets, transmembrane hydrophobic crevices of the protein, are crucial for the activation of MscL. SDSL at the nano-pockets’ entrance hinders lipid acyl chains from penetrating them and this causes the channel to adopt a more expanded state, as demonstrated by EPR spectroscopy, which is more prone to opening, as revealed by single-molecule electrophysiology. Since MscL is only found in prokaryotes, manipulation of its gating mechanism could lead to the channel being used as a novel form of antibiotic target. Moreover, due to its large pore, it could also be utilized in nanotechnological applications for targeted drug delivery.