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Study of auditory hair cells with patch clamp method

膜片钳法研究听毛细胞

基本信息

批准号：
61480104
负责人：
SUGIHARA Izumi
金额：
$ 1.86万
依托单位：
Tokyo Medical and Dental University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1986
资助国家：
日本
起止时间：
1986 至 1987
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-61480104/
关键词：
Hearing Hair cell Patch clamp goldfish Voltage clamp 膜電位固定法

项目摘要

The auditory organ of goldfish (saccule) was excised and individual hair cells were obtained by papain treatment and studied with patch clamp method.1. Transduction currents, i.e. currents responding to bending of the sensory hairs were recorded under whole-cell voltage clamp. Only eight recordings were successful yet. Technical improvements are now going on.2. Membrane potential was recorded under whole-cell current clamp. Two types of responses were observed to the square current injection. One is oscillatory change of membrane potential from 50 to 200 Hz, which was seen in ovoid or short cylindrical cells abundant in the rostral part of the saccular macula. The other is slow depolarization and action potential, which was seen in long slender cells abundant in the caudal part.3. Membrane currents were examined with voltage-clamp method under the presence of blockers and/or different ionic composition of the external solution. Ca current, Ca-activate K current, A-current, and inwardly rectifying K current were identified. In addition, transient inward current, probably Na current, was observed.4. Single channel characteristics of Ca-activated K channels (BK channels) were studied under inside-out mode. Their unitary conductance was 150 to 300 ps and it had selective permeability to K. Time course of the response to square voltage steps, measured with internal Ca concentration o 5 M was very fast as compared with similar channels in the chromaffin cell membrane.

方法1.切取金鱼听觉器官(球囊)，用木瓜酶处理获得单个毛细胞，用膜片钳方法进行研究。在全细胞电压钳下记录转导电流，即对感觉毛发弯曲作出反应的电流。到目前为止，只有8次录制成功。现在正在进行技术改进。在全细胞电流钳下记录膜电位。观察到了对方波电流注入的两种响应。一种是膜电位在50~200赫兹之间的振荡变化，这种变化见于球囊黄斑吻部丰富的卵圆形或短圆柱状细胞。另一种是慢去极化和动作电位，在尾侧部丰富的细长细胞中可见。在阻断剂和/或不同离子组成的外液存在下，用电压钳方法检测膜电流。确定了钙电流、钙激活钾电流、A电流和内向整流钾电流。此外，还观察到了瞬时内向电流，可能是钠电流。研究了自内向外模式下钙激活钾通道(BK通道)的单通道特性。它们的单位电导为150~300ps，对K具有选择性通透性。与嗜铬细胞膜上的类似通道相比，用5M测得的内钙通道对平方电压阶跃的反应时程非常快。