A Lack of Significant Lipid Interactions in the Open State of MSCS Implies a Jack-In-The-Box Type Channel Gating Mechanism

Abstract

Bacterial mechanosensitive channels are important for cell survival in changing osmotic environments. For the mechanosensitive channel of small conductance from Escherichia coli (Ec-MscS), seven residues have previously been shown to form important lipid contacts in the closed state of the channel. Based on open state crystal structures and closed state models, these residues interact with lipids in the closed state of the channel. Decreasing the hydrophobicity of these residues reduces bacterial survival upon osmotic downshock. Since the closed state appears to be stabilized by lipid interactions, we hypothesized that similar stabilizing lipid interactions could be identified in the open state. Using a computational model of open state Ec-MscS embedded in a lipid bilayer, eleven residues were determined to be lipid exposed with ten of these residues being unique to the open state of the channel. To identify the role of lipid interactions these residues were mutated to alanine and leucine to alter their interaction with the hydrophobic lipids. The effects of these mutations on channel function were assayed using osmotic downshock lysis assays, growth assays, and patch clamp electrophysiology. Mutations of the ten residues that are exposed to the lipid bilayer only in the open state of the channel effected a wildtype phenotype in all of these assays. The lack of phenotypic changes, for residues that interact with the lipid bilayer solely in the open state, suggests that these interactions are not critical for channel gating. This has led us to propose a Jack-In-The-Box model of gating for MscS, in which intrinsic lipid bilayer pressure holds the channel in the closed state. Upon relief of the intrinsic bilayer pressure by application of opposing extrinsic tension, MscS springs into the open state.