Joint Study on the Physiological Role of SA channel

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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.