Screening of SA-channel mutant in Drosophila

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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.