Machiyama Hiroaki, Tatsumi Hitoshi, Sokabe Masahiro
Department of Physiology, Nagoya University Graduate School of Medicine, Nagoya, Japan.
Biophys J. 2009 Aug 19;97(4):1048-57. doi: 10.1016/j.bpj.2009.05.021.
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.
细菌机械敏感通道MscS由跨膜（TM）结构域和大的胞质（CP）结构域组成的亚基形成同型七聚体。最近的研究表明，TM结构域的侧向扩展、CP结构域的结构变化以及TM-CP相互作用对于通道开放至关重要。然而，尚未研究CP结构域在通道开放过程中是否发生结构变化。本研究的目的是使用荧光共振能量转移（FRET）光谱法估计通道开放过程中CP结构域的结构变化。为了监测CP结构域水平直径的变化，创建了四个均具有通道活性的点突变体（A132C、F178C、L246C和R259C），并用Alexa488和Alexa568进行标记以进行FRET分析。当应用溶血磷脂酰胆碱打开通道时，这些突变体的FRET效率降低，表明通道开放时CP结构域膨胀。在缺乏TM-CP相互作用的D62N/F178C突变体中，溶血磷脂酰胆碱处理后FRET效率的降低程度小于F178C突变体。据我们所知，这些发现首次提供了实验证据，证明CP结构域在通道开放过程中膨胀，并且这种膨胀是由TM-CP相互作用介导的。
