Developments of patch-clamp technique

随着膜片钳技术的发展，普通生理学有了很大的进步。在本研究中，我们开发了几种应用膜片钳技术制备单细胞的基本方法。这些方法为该技术在生理学实验中的进一步应用提供了必要条件。1）内翻贴片的制备方法。将细胞膜暴露在空气中，制备由内而外的贴片。在不对细胞施加这种暴力的情况下，在无钙弛豫液中用微电极压碎细胞的外周部分。这打开了细胞膜，并且灌注流体容易地灌注到整个细胞中。这种由内而外的膜片膜制备方法被称为开放细胞贴附膜片法，并已用于多个实验。2）细胞内灌注法。该方法由Soejima和Noma于1983年开发，但现在已经建立并成为相当常规的方法。这种方法比原来的简单贴片方法提供了更好的千兆欧姆密封。利用这种方法，研究了细胞内ATP对钙电流的响应性、通道磷酸化的发现、细胞内酸中毒。3）细胞内物质的测定。利用对pH敏感的荧光染料，我们一直在测量单个心室肌细胞内的pH值。这笔拨款的大部分用于建造与膜片钳方法配套的分光光度计。目前，该项目正在进行试验。

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.