平面双分子层脂质膜上人工重建大电导钙激活钾通道  

Reconstitution of large conductance calcium-activated potassium channels into artificial planar lipid bilayers

作　　者：程俊[1] 曾晓荣[1] 谭晓秋[1] 李鹏云[1] 文静[1] 毛亮[1] 杨艳[1] 

机构地区：[1]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

出　　处：《生理学报》2017年第3期305-310,共6页Acta Physiologica Sinica

基　　金：supported by the National Natural Science Foundation of China(No.81173661);Joint Grant of Luzhou Municipality and Luzhou Medical College,Sichuan Province,China(No.2013LZLY-J05)

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,BK_(Ca))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N_2吹干氯仿,制成癸烷脂质液,采用涂抹法制备PLBMs,并将BK_(Ca)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BK_(Ca)的单通道的电导值为(206.8±16.9)pS,当浴液中[Ca^(2+)]=0μmol/L时,无论超极化或去极化状态下均很少见到BK_(Ca)的通道活动。膜电位为+40 m V时,当[Ca^(2+)]由1μmol/L增加到100μmol/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BK_(Ca)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BK_(Ca)通道具有电导值大、电压依赖性、Ca^(2+)敏感性和K^+选择性;(3)PLBMs技术可用于BK_(Ca)通道的药理学和动力学特性的研究。This study was aimed to establish a method to create a stable planar lipid bilayer membranes （PLBMs）, in which large con- ductance calcium-activated potassium channels （BKc,） were reconstituted. Using spreading method, PLBMs were prepared by decane lipid fluid consisting of Nz-weathered mixture of phosphatidylcholine and cholesterol at 3：1 ratio. After successful incorporation of BKca channel into PLBMs, single channel characteristics of BKc. 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 BKc~ channel activities regardless of under hyperpolarization or repolarization conditions; iv） under the condition of ＋40 mV membrane potential, BKc, 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 BKc, channels, so the PLBMs with incorporated BKCa can be used in the studies of pharmacology and dynamics of BKCa channel.

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：R3411[医药卫生—基础医学]