Joint Study on the Physiological Role of SA channel

本研究项目的目的是建立拉伸激活（SA）离子通道在生理细胞反应中的参与。所得结果如下：我们已经开发出一种方法，通过在弹性薄硅膜上培养细胞，对细胞进行定量和可重复的拉伸。通过钙显微镜测量细胞内钙的增加，可以估计内皮细胞内SA通道对机械拉伸的宏观活性。发现细胞内钙离子升高主要是由于活化的SA通道内流引起的。(2)我们开发了一种计算膜片膜和完整细胞膜张力的方法，发现激活SA通道的张力在两种膜上是相当的。先前微观（单通道）和宏观（整个细胞）观察之间的差异，其中宏观拉伸激活反应很难诱导，被认为是由于对细胞的机械拉伸效率低下造成的。(3)对培养的内皮细胞进行周期性单向拉伸，诱导细胞垂直于拉伸方向的伸长和排列。这种形态反应是由钙通过活化的SA通道内流引起的。(4)采用穿孔膜片钳法成功记录拉伸激活全细胞电流。(5)发现A6细胞对低渗刺激的细胞体积调节是由Ca通过细胞肿胀激活的SA通道内流触发的。(6)在爪蟾卵母细胞、鸡骨骼肌和心肌中，我们发现某种蜘蛛毒液能强烈阻断SA通道。我们还成功地从粗毒液中分离出部分有效成分。这可能是研究SA通道生理功能的有力工具。

The aim of this research project is to establish the involvement of stretch activated (SA) ion channels in physiological cell responses. The obtained results are as follows.(1) We have developed a method to apply quantitative and repeatable stretches to cells by culturing them on elastic thin silicon membranes. The macroscopic SA channel activities in endothelial cells to mechanical stretch could be estimated through the measurement of intracellular Ca increases by means of Ca-microscopy. It was found that the intracellular Ca increases were mainly caused by the influx through the activated SA channels.(2) We have developed a method to calculate the tension both in the patch membrane and the intact whole cell membrane and found that the tension to activate SA channels was comaparable in both membranes. The previous discrepancy between microscopic (single channel) and macroscopic (whole cell) observations, where the macroscopic stretch activated responses are very hard to induce, was thought to be caused by the infficient mechanical stretches to the cell.(3) Periodic unidirectional stretches to cultured endothelial cells induced cell elongation and alignement perpendicular to the stretch direction. This morphological response was triggered by the Ca influx through activated SA channels.(4) Stretch activated whole cell currents were successfully recorded by using the perforated patch clamp method.(5) Cell volume regulation to hypotonic stimulation in A6 cells was found to be triggered by the Ca influx through SA channels activated by cell swelling.(6) We have found a certain spider venom strongly blocked SA channels in xenopus oocytes, chick skeletal muscles, and heart muscles. We have also scceeded to make a partial purification of an active component from the crude venom. This may be a powerful tool to investigate the physiological functions of SA channels.