title: Structural changes in the cytoplasmic domain of the mechanosensitive channel MscS during opening.
abstract: The bacterial mechanosensitive channel MscS forms a homoheptamer of subunits composed of a transmembrane (TM) domain and a large cytoplasmic (CP) domain. Recent studies suggest that a lateral expansion of the TM domain, structural change in the CP domain, and TM-CP interactions are essential to open the channel. However, it has not been examined whether the CP domain undergoes structural changes during channel opening. The aim of this study was to estimate structural changes in the CP domain during channel opening using fluorescence resonance energy transfer (FRET) spectroscopy. To monitor changes in the horizontal diameter of the CP domain, four point mutants (A132C, F178C, L246C, and R259C), all of which had channel activity, were created and labeled with Alexa488 and Alexa568 for FRET analysis. The FRET efficiency of these mutants decreased when lysophosphatidylcholine was applied to open the channel, suggesting that the CP domain swells up when the channel opens. The degree of the decease in FRET efficiency after lysophosphatidylcholine treatment was smaller in the D62N/F178C mutant, which was deficient in the TM-CP interactions, than in the F178C mutant. These findings provide the first, to our knowledge, experimental evidence that the CP domain swells up during channel opening, and the swelling is mediated by the TM-CP interactions.
authors.affiliation: Department of Physiology, Nagoya University Graduate School of Medicine, Nagoya, Japan.
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Machiyama
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Tatsumi
authors.forename: Masahiro
authors.initials: M
authors.lastname: Sokabe
mesh_terms: D003593:Cytoplasm / Q000737:chemistry*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000648:ultrastructure*; D007473:Ion Channels / Q000737:chemistry* / Q000648:ultrastructure*; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D013314:Stress, Mechanical; D013329:Structure-Activity Relationship
chemical_list: D029968:Escherichia coli Proteins; D007473:Ion Channels; C469542:MscS protein, E coli
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