Screening of SA-channel mutant in Drosophila

本研究项目的目的是建立一种有效的方法来筛选缺乏拉伸激活（SA）离子通道的果蝇突变体。第一年，我们专注于建立单通道和细胞水平的筛选方法。在接下来的一年里，我们建立了果蝇胚胎骨骼肌细胞的培养体系，并在培养的细胞中发现了单个SA通道。所得结果如下：(1)通过同时测量贴片中的张力和单SA通道活性，我们可以确定激活SA通道所需的张力（5-10 dyn/cm）达到50%。我们已经在有弹性的薄硅膜上培养了细胞，通过这种膜我们可以对细胞施加定量的均匀拉伸。通过Ca显微镜测量细胞内Ca^<2+>的增加来估计拉伸诱导的SA通道活性。(3)通过模型计算，估计了硅膜细胞中SA通道激活50%时的张力。得到的张力数与膜片钳实验相当，表明硅膜法在细胞水平上是一种可靠的筛选SA通道突变体的方法。(4)建立了果蝇胚胎骨骼肌细胞培养体系。(5)在培养的肌细胞中可检测到90 pS的K^+选择性SA通道和150pS的非选择性SA通道。因此，我们建立了在单通道和细胞水平上筛选SA通道突变体的方法。利用这种方法，我们将分离果蝇有用的SA通道突变体，并克隆SA通道基因。

The aim of this research project is to establish an efficient method to screen out Drosophila mutant that lacks stretch activated (SA) ion channels. In the first year, we concentrated on establishing a screening method both at single channel and cell levels. In the following year, we established a culture system of skeletal muscle cells from Drosophila embryo, and characterized single SA channels in cultured cells. The obtained results are as follows.(1) Through simultaneous measurements of tension and single SA channel activity in the patch we could determine the tension (5-10 dyn/cm) required to activate the SA channel up to 50 %.(2) We have grown cells on elastic thin silicon membranes by which we could apply quantitative uniform stretches to the cells. The stretch-induced SA channel activity was estimated through the measurement of intracellular Ca^<2+> increases by using Ca-microscopy.(3) We made a model calculation to estimate the tension for 50 % SA channel activation in the cells on silicon membranes. The obtained number of tension was comparable to that in the patch clamp experiment, indicating that the silicon membrane method is usable as a reliable method to screen out SA channel mutant at the cell level.(4) We have established a culture system of skeletal muscle cells from Drosophila embryo.(5) We could detect a 90 pS K^+ selective SA channel and a 150pS nonselective SA channel in the cultured muscle cells.Thus, we have established the method to screen out the SA channel mutant both at the single channel and cell levels. By utilizing this method, we are going to isolate useful SA channel mutants of Drosophila and to clone SA channel genes.