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 the Escherichia coli mechanosensitive channel (MscL). Although the recent determination of the Tb-MscL crystal structure 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 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 a membrane protein can be obtained from EPR analysis of spin labeled mutants. The functional behavior of any given spin labeled mutant channel can be easily investigated by traditional electrophysiological techniques to correlate functional properties with experimentally determined structural information. MscL represents a unique model system to study the molecular basis of mechanosensation. 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.
