MscL, a prokaryotic osmolyte release valve is the best understood mechanosensitive channel. As a structurally tractable and convenient model system, it offers a unique opportunity to obtain a coherent physical picture of gating that includes the protein, surrounding lipids and water. When forced by tension, the pentameric protein expands in the plane of the membrane by about 20 nm2, opening a 3-nm aqueous pore. Thermodynamic analyses of transitions between multiple conducting states in wild-type and gain-of- function mutants strongly suggest that the transition involves a sequential action of two gates. The previously proposed model of iris-like action of the main gate has been well supported experimentally. It predicts that channel opening must be associated with a massive hydration of the inner pore surface, substantial increase of protein-lipid interface and a thickness mismatch between the protein and the lipid bilayer. Functional analysis of hydrophilic mutations in the main gate suggests that the rate-limiting step in the opening pathway is likely to be the critical event of the pore wetting^ Recently MscL was found to be cold-activated and there are indications that perturbations in the surrounding bilayer change the slope of the temperature dependence. The proposal addresses two main questions: what is the contribution of pore hydration to the energetics and kinetics of gating, and could the conformational state of boundary lipids confer steep temperature sensitivity? To resolve these important details of the channel mechanism, we propose: (1) molecular dynamics simulations of wetting/dewetting processes in the course of pore expansion with additional thermodynamic calculations of interfacial energies for the protein surface and 'vapor plug'; (2) experimental patch-clamp analysis of mutants with altered hydration properties of the pore, including thermodynamic cycles with protonated and de-protonated histidines; (3) experimental studies of MscL temperature dependencies and (4) computational analysis of perturbations of the lipid bilayer by the channel protein in different conformations. Implementation of this research plan promises to clarify the general principles of a hydrophobic gate action and the role of the lipid environment in setting temperature dependencies for sensory channels. ? ? ?
