Membrane stiffness is a key determinant of E coli MscS channel mechanosensitivity

Prokaryotic mechanosensitive (MS) channels have an intimate relationship with membrane lipids. Membrane lipids may influence channel activity by directly interacting with bacterial MS channels or by influencing the global properties of the membrane such as area stretch and bending moduli. Previous work has implicated membrane stiffness as a key determinant of the mechanosensitivity of E. coli (Ec)MscS. Here we systematically tested this hypothesis using patch fluorometry of azolectin liposomes doped with lipids of increasing area stretch moduli. Increasing DOPE content of azolectin liposomes causes a rightward shift in the tension response curve of EcMscS. These rightward shifts are further magnified by the addition of stiffer forms of PE such as the branched chain lipid DPhPE and the fully saturated lipid DSPE. Furthermore, a comparison of the branched chain lipid DPhPC to the stiffer DPhPE showed a rightward shift in the tension response curve in the presence of the stiffer DPhPE. We show that these changes are not due to changes in membrane bending rigidity as the tension threshold of EcMscS in membranes doped with PC18:1 and PC18:3 are the same, despite a two-fold difference in their bending rigidity. We also show that after prolonged pressure application sudden removal of force in softer membranes causes a rebound reactivation of EcMscS and we discuss the relevance of this phenomenon to bacterial osmoregulation. Collectively, our data demonstrate that membrane stiffness is a key determinant of the mechanosensitivity of EcMscS.