Sensing mechanical forces is crucial for the survival of prokaryotic and eukaryotic cells. Mechanosensation is used in the senses of touch and balance as well as blood pressure and osmo-regulation. One of the primary sensors employed across species is the mechanosensitive (MS) channel. Even bacteria express MS channels, which play the role of a biological "emergency release valve" upon acute osmotic stresses. MscL and MscS are two independent families of MS channels best studied in E. coli, but found across the bacterial kingdom. Previous studies have demonstrated that these channels, as well as at least some eukaryotic MS channels, appear to directly sense and respond to tension in the lipid bilayer;thus protein-lipid interactions are important for this class of ion channels. Here w propose to examine the protein-lipid interactions important for MscS sensing and responding to membrane tension. MscS is an ideal system for studying functional protein-lipid interactions because the wild type or mutant MscS protein can be purified and reconstituted into various defined lipid environments and assayed by patch clamp. Initially we will determine if specific amino acids of MscS transduce membrane tension through direct interactions with lipid headgroups. Specifically, using rapid in vivo assays developed in the Blount laboratory we will screen selected point mutations to determine if they lead to channels that are more or less sensitive to membrane tension;this subset of mutants will be further characterized by patch clamp. We will also determine which of the lipid headgroups are important for these interactions by reconstituting the MscS channel in membranes made of various lipids. Hypotheses of specific residue/headgroup interactions will then be tested by combining mutant proteins into the defined lipid membranes. Tryptophan fluorescence will be used to verify the relative affinities of various lipid headgroups for specific residues of MscS. Finally, we will determine if hydrophobic mismatch serves as a stimulus for MS Channel gating. To achieve this goal I will reconstitute both Wt and mutated channels into lipids with various carbon tail lengths. By creating variations in the membrane thickness I will be changing the environment for residues at the protein lipid interface. The effects of these variations will be measured using tryptophan fluorescence and single channel analysis. The results of these studies will provide insight into how membrane tension is transuded to a mechanosensor to change it conformational structure.
MscL and MscS share a similarity to mammalian mechanosensitive (MS) channels in that they appear to be able to sense and respond to biophysical changes in their lipid environment, thus protein-lipid interactions are of critical importance. Bacteria currently provide a more tractable system for studying MS channel function because the channels can be functionally reconstituted into defined lipids. Using mutagenesis and such reconstitution, this project aims at determining how one of these sensors, MscS, functionally interacts with its membrane environment.