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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM063617-05
Application #
6988715
Study Section
Biochemistry and Biophysics of Membranes Study Section (BBM)
Program Officer
Shapiro, Bert I
Project Start
2001-07-01
Project End
2005-11-30
Budget Start
2005-07-01
Budget End
2005-11-30
Support Year
5
Fiscal Year
2005
Total Cost
$129,794
Indirect Cost
Name
University of Virginia
Department
Physiology
Type
Schools of Medicine
DUNS #
065391526
City
Charlottesville
State
VA
Country
United States
Zip Code
22904
Bavi, Navid; Martinac, Adam D; Cortes, D Marien et al. (2017) Structural Dynamics of the MscL C-terminal Domain. Sci Rep 7:17229
Bavi, Navid; Cortes, D Marien; Cox, Charles D et al. (2016) The role of MscL amphipathic N terminus indicates a blueprint for bilayer-mediated gating of mechanosensitive channels. Nat Commun 7:11984
Vasquez, Valeria; Sotomayor, Marcos; Cortes, D Marien et al. (2008) Three-dimensional architecture of membrane-embedded MscS in the closed conformation. J Mol Biol 378:55-70
Vasquez, Valeria; Sotomayor, Marcos; Cordero-Morales, Julio et al. (2008) A structural mechanism for MscS gating in lipid bilayers. Science 321:1210-4
Vasquez, Valeria; Cortes, D Marien; Furukawa, Hiro et al. (2007) An optimized purification and reconstitution method for the MscS channel: strategies for spectroscopical analysis. Biochemistry 46:6766-73
Sotomayor, Marcos; Vasquez, Valeria; Perozo, Eduardo et al. (2007) Ion conduction through MscS as determined by electrophysiology and simulation. Biophys J 92:886-902
Denmark, Scott E; Baird, John D (2006) Palladium-catalyzed cross-coupling reactions of silanolates: a paradigm shift in silicon-based cross-coupling reactions. Chemistry 12:4954-63