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

Abstract

41Prokaryotic mechanosensitive (MS) channels have an intimate relationship with 42 membrane lipids. Membrane lipids may influence channel activity by directly 43 interacting with bacterial MS channels or by influencing the global properties of the 44 membrane such as area stretch and bending moduli. Previous work has implicated 45 membrane stiffness as a key determinant of the mechanosensitivity of E. coli (Ec)MscS. 46 Here we systematically tested this hypothesis using patch fluorometry of azolectin 47 liposomes doped with lipids of increasing area stretch moduli. Increasing DOPE 48 content of azolectin liposomes causes a rightward shift in the tension response curve of 49 EcMscS. These rightward shifts are further magnified by the addition of stiffer forms 50 of PE such as the branched chain lipid DPhPE and the fully saturated lipid DSPE. 51 Furthermore, a comparison of the branched chain lipid DPhPC to the stiffer DPhPE 52 showed a rightward shift in the tension response curve in the presence of the stiffer 53DPhPE. We show that these changes are not due to changes in membrane bending 54 rigidity as the tension threshold of EcMscS in membranes doped with PC18:1 and 55 PC18:3 are the same, despite a two-fold difference in their bending rigidity. We also 56 show that after prolonged pressure application sudden removal of force in softer 57 membranes causes a rebound reactivation of EcMscS and we discuss the relevance of 58 this phenomenon to bacterial osmoregulation. Collectively, our data demonstrate that 59 membrane stiffness is a key determinant of the mechanosensitivity of EcMscS. 60 61 62 65 This channel is the canonical member of a diverse family of MS channels that spans 66 prokaryotic and eukaryotic cell-walled organisms 3-5 . Purification and reconstitution of 67EcMscS, and many of its homologues, into lipid bilayers show that it gates according 68 to the force-from-lipid principle 6-8 . This means the channel is inherently 69 mechanosensitive and directly senses membrane forces that result in a conformational 70 change culminating in the channel opening. As a result, it is clear that membrane lipids 71 are a key driver of EcMscS activity. Recent evidence suggests that eukaryotic MS 72 channels also employ force-from-lipids gating 9-12 . Therefore, the basic biophysical 73 principles that govern the gating of prokaryotic channels may in turn provide insight 74 into the gating of eukaryotic MS channels 13, 14 . 76Liposomal reconstitution has for many years been successfully used not only to 77 document the inherent mechanosensitivity of both prokaryotic and eukaryotic ion 78 channels but also to probe the influence of individual lipids on MS channel function 15-79 17 . Lipids can influence integral membrane proteins such as MS channels in one of two 80 ways 18 . Firstly, the lipid may directly interact with the protein and modify function. The 81 second is that lipids may indirectly affect function via global effects on the mechanical 82 properties of the bilayer. For example, liposomal reconst...

Approaches for the modulation of mechanosensitive MscL channel pores

“…Modulators can act indirectly on MS channels, such as through the modification of the physical or chemical properties of the membrane. A decrease in membrane thickness was shown to lower the activation threshold of EcMscL, while decreased membrane fluidity hampered EcMscS gating (Perozo et al, 2002;Nomura et al, 2012;Xue et al, 2020). An atomistic MD study showed that the thickness-dependent gating of MscL is likely to be driven by the hydrophobic matching of the protein to the thickness of the bilayer, largely through the interaction of F78 (TbMscL) with the membrane surface on TM2 (Katsuta et al, 2019).…”