Biophysical and Molecularbiological Study on Stretch Channel

本课题旨在建立一种分析拉伸激活（SA）离子通道中刺激-反应关系的方法，并获得分离SA通道分子的线索。得到的结果如下：我们已经发展了一种理论来分析贴片区域对拉伸脉冲的瞬态响应，通过它我们可以确定贴片的机械性能，如弹性或粘滞性。(2)通过上述理论分析，我们发现大多数具有gigasseal的膜片膜在密封形成过程中可能产生相当大的静息张力（约5dyn/cm）。这一结果非常重要，因为它可以解决微观（单通道）和宏观（整个细胞）观察之间的矛盾，其中宏观拉伸激活反应很难诱导（Morris & Horn, 1991）。我们已经开发出一种方法，通过在弹性薄硅膜上生长细胞，将可控制的拉伸施加到整个细胞上。此外，我们可以通过使用Ca显微镜测量细胞内Ca^<2+>的增加，成功地估计全细胞SA通道的活性。(4)取贴片的静息张力，发现激活贴片中SA通道的张力与完整细胞膜的张力相当，这是用(3)中描述的方法测量的。这一结果有力地支持了这样一种观点，即通过贴片夹紧观察到的单SA通道活性参与了宏观拉伸依赖的细胞反应。(5)我们发现了有效的SA通道阻滞剂和激活剂。其中一些对今后SA通道分子的鉴定和分离具有一定的参考价值。

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.