Developments of patch-clamp technique

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General physiology has been enormously progressed following the development of the patch clamp technique. Here in this grant, we developed several basic methods for the application of the patch clamp techique to a single cell preparation. These methods were necessary for the further application of the thechnique for the physiological experiment.1) Method of preparing inside out patch. Inside out patch has been prepared by exposing the cell membrane within air. Without such a violence to the cell, the peripheral part of the cell was crushed with microelectrode within Ca-free relaxation fluid. This opens the cell membrane and the perfusion fluid perfused throughout the cell easily. This method of preparing the inside out patch membrane was named as the open cell attached patch method and has been used in several experiments.2) Intracellular perfusion method. This method has been developed by Soejima and Noma in 1983, but now has been established and became rather rutine method. This method provides better giga-ohm seal than the original simple patch method. Using this method, responsibility of intracellular ATP on the calcium current, discovery of the phosphorylation of the channel, intracellular acidosis have been investigated.3) Measurement of the intracellular substances. Using fluorescent dyes sensitive to pH, we have been measuring the intracellular pH of the single ventricular cell. Major part of this grant has been spent for constructing spectrophotometric devises accompanying with the patch clamp method. Experiments are now progressing on this project.