Biophysical and Molecularbiological Study on Stretch Channel

本课题的目的是建立一种分析牵张激活（stretchactivated，SA）离子通道的刺激-反应关系的方法，并为SA通道分子的分离提供线索。所得结果如下。(1)我们发展了一种理论来分析贴片区域对拉伸脉冲的瞬态响应，通过该理论我们可以确定贴片的力学性质，如弹性或粘性。(2)通过上述理论分析，我们发现大多数含有千兆密封的补片膜都有相当大的静息张力（约为1000）。5dyn/cm），可能在密封形成过程中产生。这一结果非常重要，因为它可以解决微观（单通道）和宏观（全细胞）观察之间的矛盾，其中宏观拉伸激活反应很难诱导（Morris & Horn，1991）。(3)我们已经开发出一种方法，通过在弹性薄硅膜上生长细胞来对整个细胞施加可控的拉伸。此外，我们可以通过使用钙显微镜测量细胞内Ca^<2+>的增加来成功地估计整个细胞SA通道的活性。(4)取膜片中的静息张力，发现膜片中激活SA通道的张力与完整细胞膜中的张力相当，其通过（3）中描述的方法测量。这一结果有力地支持了这样的想法，即通过膜片钳观察到的单个SA通道活性参与宏观拉伸依赖性细胞反应。(5)我们已经发现了SA通道的有效阻断剂和激活剂。其中一些结果对今后SA通道分子的鉴定和分离有一定的参考价值。

The aim of this research project is to establish a method for the analysis of the stimulusresponse relationship in the stretch activated (SA) ion channel and to obtain a clue to isolate SA channel inolecules. The obtained results are follows.(1) We have developed a theory to analyze transient response of the patch area to a stretch pulse, by which we could determine the mechanical property, like elasticity or viscocity, of the patch.(2) Through the analysis by using above theoty, we found that most patch membranes with gigaseal have considerable amount of resting tension (ca. 5dyn/cm), that might generate during seal formation. This result is very important, because it can resolve the contradiction between microscopic (single channel) and macroscopic (whole cell) observations, where the macroscopic stretch activated responses are very hard to induce (Morris & Horn, 1991).(3) We have developed a method to apply controllable stretches to the whole cell by growing cells on elastic thin silicon membranes. In addition we could successfully estimate whole cell SA channel activity through the measurement of intracellular Ca^<2+> increases by using Ca-microscopy.(4) Taking the resting tension in the patch, the tension to activate SA channels in the patch was found to be comparable to that in intact cell membranes, which was measured by the method described in (3). This result strongly support the idea that the single SA channel activity observed by the patch clamping is involved in macroscopic stretchdependent cell responses.(5) We have found potent blockers and activatiors for the SA channel. Some of them should be useful for the future study to identify and isolate SA channel molecules.