Developments of patch-clamp technique

膜片钳技术的发展

• 批准号: 60870004
• 负责人: IRISAWA Hiroshi
• 金额: $ 5.76万
• 依托单位: Okazaki National Research Institutes
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Developmental Scientific Research
• 财政年份: 1985
• 资助国家: 日本
• 起止时间: 1985 至 无数据
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-60870004/
• 关键词: patch clamp method; open cell inside out patch; intracellular perfusion; INSIDE; OUTパッチ法の改良

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.

随着膜片钳技术的发展，普通生理学已经取得了巨大的进步。在这项资助中，我们开发了几种将膜片钳技术应用于单细胞制备的基本方法。这些方法为该技术在生理实验中的进一步应用奠定了基础。1)内外贴片的制备方法。通过将细胞膜暴露在空气中来制备由内到外的贴片。在没有这种对细胞的暴力作用的情况下，细胞的外围部分被微电极在无ca松弛液中粉碎。这打开了细胞膜，灌注液很容易在整个细胞中灌注。这种制备内外贴片膜的方法被命名为开细胞贴片法，并已在若干实验中得到应用。2)细胞内灌注法。这种方法是由Soejima和Noma在1983年开发的，但现在已经确立并成为一种相当常规的方法。这种方法比原始的简单贴片方法提供更好的千兆欧姆密封。利用这种方法，研究了细胞内ATP对钙电流的作用，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值敏感的荧光染料，我们一直在测量单个心室细胞的细胞内pH值。这笔拨款的主要部分用于构建伴随膜片钳法的分光光度仪。这个项目的实验正在进行中。

项目成果

Circulation Research. 57. 553-561 (1985)

流通研究。

Circ Res.57. (1985)

循环第 57 号决议。

Circulation Research. 57. 65-73 (1985)

流通研究。