Bartlett Jessica L, Li Yuezhou, Blount Paul
Department of Physiology, University of Texas-Southwestern Medical Center, Dallas, Texas, USA.
Biophys J. 2006 Nov 15;91(10):3684-91. doi: 10.1529/biophysj.106.088062. Epub 2006 Aug 25.
The mechanosensitive channel of large conductance acts as a biological "emergency release valve" that protects bacterial cells from hypoosmotic stress. Although structural and functional studies and molecular dynamic simulations of this channel have led to several models for the structural transitions that occur in the gating process, inconsistencies linger and details are lacking. A previous study, using a method coined as the "in vivo SCAM", identified several residues in the channel pore that were exposed to the aqueous environment in the closed and opening conformations. Briefly, the sulfhydryl reagent MTSET was allowed to react, in the presence or absence of hypoosmotic shock, with cells expressing mechanosensitive channel of large conductance channels that contained cysteine substitutions; channel dysfunction was assessed solely by cell viability. Here we evaluate the MTSET-induced functional modifications to these mechanosensitive channel activities by measuring single channel recordings. The observed changes in residue availability in different states, as well as channel kinetics and sensitivity, have allowed us to elucidate the microenvironment encountered for a number of pore residues, thus testing many aspects of previous models and giving a higher resolution of the pore domain and the structural transitions it undergoes from the closed to open state.
大电导机械敏感通道充当生物“应急释放阀”，保护细菌细胞免受低渗胁迫。尽管对该通道的结构和功能研究以及分子动力学模拟已得出几种门控过程中发生的结构转变模型，但仍存在不一致之处且细节缺失。先前一项研究使用一种名为“体内扫描诱变”的方法，确定了通道孔中在关闭和开放构象下暴露于水环境的几个残基。简要来说，在有或无低渗休克的情况下，让巯基试剂MTSET与表达含有半胱氨酸替代的大电导机械敏感通道的细胞反应；仅通过细胞活力评估通道功能障碍。在这里，我们通过测量单通道记录来评估MTSET对这些机械敏感通道活性的功能修饰。在不同状态下观察到的残基可用性变化以及通道动力学和敏感性，使我们能够阐明许多孔残基所遇到的微环境，从而检验先前模型的许多方面，并给出孔结构域及其从关闭状态到开放状态所经历的结构转变的更高分辨率。
