Mechanosensitive (MS) channels are oligomeric membrane proteins that respond to changes in bilayer tension by catalyzing the transfer of ions and other solutes across the membrane, fulfilling a major role in the response of living organisms to mechanical stimuli. These channels are considered to function as mechano-electrical switches in such diverse physiological processes as touch, hearing, proprioception, turgor control in plant cells and osmoregulation in bacteria. The overall, long-term goal of this project is to understand the molecular mechanism of gating in prokaryotic mechanosensitive channels. Although the recent determination of the MscL and MscS crystal structures has dramatically improved our knowledge of this class of molecules, a number of mechanistic questions remain to be solved. This is particularly true for the molecular events underlying channel gating. In this respect, we plan to experimentally address several fundamental questions: What regions of the channel form the gate(s) and how do they move to produce gating? What is the physical basis of the energy transduction steps, starting with transbilayer tension and culminating in protein motion? Where in the molecule does mechanical transduction occur? What are the structures of the key functional states? The fact that MscL and MscS can be activated by pressure gradients both in native membrane and after reconstitution in pure lipid systems indicates that these channels are gated directly by tension transmitted through the bilayer. Therefore, establishing the physical principles underlying the energy transduction steps in these proteins will require studying the role of protein-lipid interactions in this process. The approach we plan to pursue combines reporter-group spectroscopic techniques (spin labeling/EPR, Fluorescence) and electrophysiological methods with classical biochemical and molecular biological procedures. Functional studies will be targeted to understand the physical basis of energy transduction in mechanosensitive channels. Information on the topology, secondary, and tertiary structure of MscS and MscL will be obtained from EPR analysis of spin labeled mutants. The data will be interpreted to generate backbone models of the different stages of the gating pathway in each type of channel. This proposal opens up a new experimental avenue that will contribute to the understanding of biologically important events such as ion channel gating, nociception and signal transduction.
