论文部分内容阅读
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。
The purpose of this study was to explore a method for the preparation of a stable planar bilayer membranes (PLBMs) and attempt to establish a large conductance calcium-activated potassium channel (BK_ (Ca) ) Protein fusion on which a single-channel current study was performed. The lecithin and cholesterol chloroform were mixed in a ratio of 3: 1, and the chloroform was dehydrated by N 2 to make decane lipid solution. PLBMs were prepared by smear method and BK_ (Ca) was reconstituted into PLBMs. Single channel characteristics. The results showed that the conductivity of single channel of BK_ (Ca) reconstituted to PLBMs was (206.8 ± 16.9) pS. When [Ca 2+] = 0μmol / L in the bath, BK_ (Ca) channel activity is seldom seen in the polarized state. When the membrane potential was +40 mV, the channel open probability increased from 0.034 ± 0.002 to 0.961 ± 0.013 [Ca 2+] increased from 1μmol / L to 100μmol / L, and the average open time increased from (1.1 ± 0.2 ) was prolonged to (151.3 ± 10.2) ms and the mean closure time was shortened from (32.2 ± 2.8) ms to (2.1 ± 1.8) ms. When K + concentration was 40/140 mmol / L (Ca) reversal potential at -30 mV or so. The above results suggest that: (1) smear method can be used for the preparation of PLBMs; (2) the reconstructed BK Ca channel has the characteristics of large conductance, voltage dependence, Ca 2+ sensitivity and K + selectivity; (3) PLBMs technology can be used to study the pharmacological and kinetic properties of BK_ (Ca) channels.