动脉平滑肌细胞膜片制作及其大电导钙激活钾通道的特性

目的: 探讨人体小动脉平滑肌细胞膜片制作方法,观察其上大电导钙激活钾通道(large-conductance calcium-activatedpotassium channels,BK_Ca)的特性。方法: 将新鲜分离出的人肠系膜动脉,分离出平滑肌层,经酶解得单个平滑肌细胞。取平滑肌细胞置于含对称性高钾(K+140 mmol/L)浴液中,用膜片钳技术记录其单通道电流,输入计算机进行数据采集与分析。观察动脉平滑肌细胞三种膜片的制作过程和BK_Ca的特性。结果: 在肠系膜动脉平滑肌细胞膜上分别形成了细胞贴附式、内面向外式、全细胞记录三种膜片。用膜片钳技术观察到急性酶分离的人体肠系膜动脉平滑肌细胞上BK_Ca通道具有钙敏感性:表现为胞内游离钙离子浓度增加,通道开放事件数目增加;电压依赖性:表现为随着膜电位的增加,通道开放事件数逐渐增加;电导值大:本实验的电导值为198.6±38.09 pS;应用TEA+为50 mmol/L时通道活动完全阻断。结论: 人体肠系膜动脉平滑肌细胞上BK_Ca通道具有钙敏感性、电压依赖性、大电导、TEA+敏感特性。

Processing of artery smooth muscle cells patch and the properties of largeconductance calcium-activated potassium channels

Objective: To investigate the processing of the artery smooth muscle cells patch and to observe the properties of large-conductance calcium-activated potassium channels in them.Methods: The humans mesenteric artery was dissociated from human mesenteriolum,and single smooth muscle cells were isolated by enzyme and put into symmetric high potassium solution.The channel current was recorded by using patch clamp and the data were analyzed by the computer.The processing of three kinds of ptches of artery smooth muscle cell and the characteristics of BK_Ca were observed.Results: In the experiment,cell attached,inside-out and whole cell recording patches were produced respectively on the cytomembrane of mesenteric artery smooth muscle.BK_Ca channels of smooth muscle cells isolated by enzyme were sensitive toCa2+]i and dependant on voltage;the channels opened more frequently with the increase of freeCa2+]i and membrane potential.The conductance of enzymatic smooth muscle cells in this experiments was 198.6±38.09 pS;The channels were sensitive to TEA+:when 50 mmol/L TEA+ was used,these channels were entirely blocked.Conclusions: The experiments suggested that BK_Ca channels in smooth muscle cells isolated by enzyme were sensitive to Ca2+,dependent of voltage sensitive to TEA+ and had the properties of large conductance.