Article

An in vivo assay identifies changes in residue accessibility on mechanosensitive channel gating.

Department of Physiology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390-9040, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.81). 08/2004; 101(27):10161-5. DOI: 10.1073/pnas.0402040101
Source: PubMed

ABSTRACT MscL is a mechanosensitive channel of large conductance that functions as an "emergency release valve," allowing bacteria to survive acute hypoosmotic stress. Although Escherichia coli MscL is the best-studied mechanosensitive channel, structural rearrangements occurring during gating remain disputed. Introduction of a charged residue into the pore of MscL was shown to result in a reduced-viability phenotype. Here, we probe for residues in the transmembrane domains that are exposed to the aqueous environment in the presence and absence of hypoosmotic shock by reacting a charged sulfhydryl reagent with substituted cysteines. Subsequent analysis of cell viability allows for an assessment of residues exposed in the closed and opening states in vivo. The results suggest that the crystal structure of MscL derived from the Mycobacterium tuberculosis orthologue may reflect a nearly closed rather than fully closed state and support a clockwise rotation of the pore-forming first transmembrane domain on gating.

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