过刊浏览

平面双分子层脂质膜上人工重建大电导钙激活钾通道

程俊, 曾晓荣, 谭晓秋, 李鹏云, 文静, 毛亮, 杨艳^*

西南医科大学心血管医学研究所，心血管疾病预防和治疗协同创新中心，医学电生理学教育部重点实验室，泸州 646000

摘要

本研究旨在探索建立一种稳定平面双分子层脂质膜(planar lipid bilayer membranes, PLBMs)的制备方法，并尝试将大电导钙激活钾通道(large conductance calcium-activated potassium channel, BKCa)蛋白融合其上进行单通道电流研究。将卵磷脂与胆固醇氯仿按3:1比例混合，用N2吹干氯仿，制成癸烷脂质液，采用涂抹法制备PLBMs，并将BKCa重建到PLBMs上，用膜片钳研究其单通道特性。结果显示，重建到PLBMs上的BKCa的单通道的电导值为(206.8 ±16.9) pS，当浴液中[Ca2+] = 0 μmol/L时，无论超极化或去极化状态下均很少见到BKCa的通道活动。膜电位为+40 mV时，当[Ca2+]由1 μmol/L增加到100 μmol/L时，通道开放概率由0.034 ± 0.002增加到0.961 ± 0.013，平均开放时间由(1.1 ± 0.2) ms延长到(151.3 ± 10.2) ms，平均关闭时间由(32.2 ± 2.8) ms缩短为(2.1 ± 1.8) ms；当K+浓度为40/140 mmol/L (Cis/Trans)时，重建的BKCa反转电位在−30 mV左右。以上结果提示：(1) 涂抹法可用于PLBMs的制备；(2) 重建后的BKCa通道具有电导值大、电压依赖性、Ca2+敏感性和K+选择性；(3) PLBMs技术可用于BKCa通道的药理学和动力学特性的研究。

关键词： 平面双分子层脂质膜; 涂抹法; 大电导钙激活钾通道

分类号：R33-3

[Reconstitution of large conductance calcium-activated potassium channels into artificial planar lipid bilayers.] [Article in Chinese]

Cheng Jun, ZENG Xiao-Rong, TAN Xiao-Qiu, LI Peng-Yun, WEN Jing, MAO Liang, YANG Yan^*

Key Laboratory of Medical Electrophysiology of Ministry of Education, Collaborative Innovation Center for Prevention and Treatment of Cardiovascular Disease, Institute of Cardiovascular Research, Southwest Medical University, Luzhou 646000, China

Abstract

This study was aimed to establish a method to create a stable planar lipid bilayer membranes (PLBMs), in which large conductance calcium-activated potassium channels (BKCa) were reconstituted. Using spreading method, PLBMs were prepared by decane lipid fluid consisting of N2-weathered mixture of phosphatidylcholine and cholesterol at 3:1 ratio. After successful incorporation of BKCa channel into PLBMs, single channel characteristics of BKCa were studied by patch clamp method. The results showed that i) the single channel conductance of BKCa was (206.8 ± 16.9) pS; ii) the activities of BKCa channel were voltage dependent; iii) in the bath solution without Ca2+, there was almost no BKCa channel activities regardless of under hyperpolarization or repolarization conditions; iv) under the condition of +40 mV membrane potential, BKCa channels were activated in a Ca2+ concentration dependent manner; v) when [Ca2+] was increased from 1 μmol/L to 100 μmol/L, both the channel open probability and the average open time were increased, and the average close time was decreased from (32.2 ± 2.8) ms to (2.1 ± 1.8) ms; vi) the reverse potential of the reconstituted BKCa was −30 mV when [K+] was at 40/140 mmol/L (Cis/Trans). These results suggest that the spreading method could serve as a new method for preparing PLBMs and the reconstituted BKCa into PLBMs showed similar electrophysiological characteristics to natural BKCa channels, so the PLBMs with incorporated BKCa can be used in the studies of pharmacology and dynamics of BKCa channel.

Key words: planar bilayer lipid membrane; spreading method; large conductance calcium-activated potassium channel

收稿日期：2016-12-08　　录用日期：2017-02-05

通讯作者：杨艳　　E-mail: wyangyan@wsmu.edu.cn

引用本文：

程俊, 曾晓荣, 谭晓秋, 李鹏云, 文静, 毛亮, 杨艳. 平面双分子层脂质膜上人工重建大电导钙激活钾通道[J]. 生理学报 2017; 69 (3): 305-310.

Cheng Jun, ZENG Xiao-Rong, TAN Xiao-Qiu, LI Peng-Yun, WEN Jing, MAO Liang, YANG Yan. [Reconstitution of large conductance calcium-activated potassium channels into artificial planar lipid bilayers.] [Article in Chinese]. Acta Physiol Sin 2017; 69 (3): 305-310 (in Chinese with English abstract).