STUDY ON THE STRUCTURE FUNCTION AND PHYSIOLOGICAL ROLES OF SA CHANNELS

本研究的目的有三个方面：1)阐明已知结构的SA通道的结构功能；2)从高等生物中鉴定SA通道的分子结构；3)阐明SA通道和细胞骨架在机械诱导的细胞信号传导中的作用。主要结果如下。(1)细菌SA通道MscL机械敏感结构域的鉴定：我们发现位于脂质双分子层质周边界的疏水氨基残基可作为MscL的机械传感器。这个位点对应于脂质甘油主链，这是最适合感知膜张力的结构。(2)心脏SA通道SAKCA的分子鉴定：从鸡心脏中克隆了一个拉伸激活的大Kca通道的编码基因，并在c端鉴定了59个氨基酸序列STREX作为该通道的机械敏感结构域。我们还分离出一个50 KDa的蛋白作为辅助装置，将膜上的张力传递给STREX。(3)细胞骨架在SA通道激活中的作用：我们发现应力纤维在SA通道激活中起着力传递的作用。(4) SA通道在细胞信号传导中的作用：我们分析了在机械刺激下导致NF-kB激活的细胞内信号传导。提示细胞可以根据机械刺激的时间结构选择不同的机械信号级联。对周期性或短暂性机械刺激的机械信号主要由SA通道介导，而对持续性机械刺激的机械信号主要由整合素介导

The aim of this study was three fold : 1) elucidation of structure function of SA channels with known structure, 2) molecular identification of SA channels from higher order organisms, and 3) elucidation of roles of SA channels and cytoskeletons in mechano-induced cell signaling. Major results are as follows. (1) Identification of mechano-sensitive domain of the bacterial SA channel MscL : We found that hydrophobic amino-residues located at the peri-plasmic boundary of the lipid bilayer act as a mechanosensor of MscL. This site corresponds to the lipid glycerol backbone that is the most suitable structure for sensing tension in the membrane. (2)Molecular identification of the heart SA channel SAKCA : We cloned a gene encoding a stretch activated big Kca channel from chick heart and identified the 59 amino acids sequence called STREX at C-terminus as a mechanosensitive domain of this channel. We also isolated a 50 KDa protein acting as an accessory device that conveys tension in the membrane to STREX. (3) Role of cytosekelton in SA channel activation : We found that the stress fiber acts as a force transmitter in SA channel activation. (4) Role of SA channel in cell signaling : We analyzed the intracellular signaling leading to NF-kB activation in response to mechanical stimuli. It is suggested that cells can chose different mechano-signaling cascade depending on the time structure of mechanical stmuli. Mechano-signaling to cyclic or transient mechanical stimuli is mainly mediated by SA channels, while those to constant mechanical stimuli by integrin