The small mechanosensitive channel MscS, a ubiquitous bacterial osmoregulator, is an advanced model system for biophysical studies of the initial events in mechanotransduction. The solved crystal structure and the existence of eukaryotic homologs make MscS especially attractive. Our preliminary data, both experimental and computational, lay the foundation for a new hypothesis about the gating mechanism of MscS which we now present as a series of conformational states and transitions derived from the crystal structure. Despite previous notions that MscS is a tension and voltage-activated channel, we found its activation by tension rather voltage-independent. However, the process of inactivation was strongly promoted by depolarization. Computational assessment of the crystal structure suggested that the pore is dehydrated and its conformation represents a non-conducting, likely inactivated state. Using targeted energy minimizations we have envisioned a gating cycle which begins with a compact resting conformation of the barrel with transmembrane helices tightly packed around the pore. Opening is achieved through a concerted outward movement of helices associated with wetting and expansion of the pore constriction. Inactivation occurs when the pore-forming TM3 helices decouple from the lipid-facing TM1 and TM2 helices and collapse into a narrow (crystal-like) conformation. To test this hypothesis we will (1) perform steered molecular dynamics simulations and generate accurate models for the closed and open states; (2) verify the predicted proximities of critical residues by disulfide cross-linking and test the functional consequences of bridge formation in patch-clamp experiments; (3) test accessibilities of residues in the pore and crevices using cysteine substitutions and MTS reagents; (4) evaluate the contribution of the pore hydration to the gating energetics and validate the previously proposed Vapor lock' mechanism. The work, when accomplished, will move us closer toward understanding the mechanisms of the growing families of sensory channels. ? ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM075225-01A1
Application #
7105290
Study Section
Biochemistry and Biophysics of Membranes Study Section (BBM)
Program Officer
Shapiro, Bert I
Project Start
2006-03-01
Project End
2010-02-28
Budget Start
2006-03-01
Budget End
2007-02-28
Support Year
1
Fiscal Year
2006
Total Cost
$282,150
Indirect Cost
Name
University of Maryland College Park
Department
Biology
Type
Schools of Earth Sciences/Natur
DUNS #
790934285
City
College Park
State
MD
Country
United States
Zip Code
20742
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Sukharev, Sergei; Sachs, Frederick (2012) Molecular force transduction by ion channels: diversity and unifying principles. J Cell Sci 125:3075-83
Kamaraju, Kishore; Smith, Jacqueline; Wang, Jingxin et al. (2011) Effects on membrane lateral pressure suggest permeation mechanisms for bacterial quorum signaling molecules. Biochemistry 50:6983-93
Kamaraju, Kishore; Belyy, Vladislav; Rowe, Ian et al. (2011) The pathway and spatial scale for MscS inactivation. J Gen Physiol 138:49-57
Boer, Miriam; Anishkin, Andriy; Sukharev, Sergei (2011) Adaptive MscS gating in the osmotic permeability response in E. coli: the question of time. Biochemistry 50:4087-96
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Anishkin, A; Akitake, B; Kamaraju, K et al. (2010) Hydration properties of mechanosensitive channel pores define the energetics of gating. J Phys Condens Matter 22:454120
Belyy, Vladislav; Kamaraju, Kishore; Akitake, Bradley et al. (2010) Adaptive behavior of bacterial mechanosensitive channels is coupled to membrane mechanics. J Gen Physiol 135:641-52
Kung, Ching; Martinac, Boris; Sukharev, Sergei (2010) Mechanosensitive channels in microbes. Annu Rev Microbiol 64:313-29
Anishkin, Andriy; Sukharev, Sergei (2009) State-stabilizing Interactions in Bacterial Mechanosensitive Channel Gating and Adaptation. J Biol Chem 284:19153-7

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