表达在HEK293细胞上的BK_(Ca)通道基本特性与动力学调控研究

Basic characteristics and kinetic regulation of BKCa expressed in HEK 293 cell lines

目的构建稳定表达人血管平滑肌大电导钙激活钾通道(BKCa)的293细胞系,并研究其基本电生理学特征和动力学模型。方法通过脂质体(lipofectamine 2000)转染法转染BKCa表达质粒pcDNA3.1-BKα和(或)pIRES-BKβ1,筛选稳定表达的单克隆细胞系。使用膜片钳多种记录方式研究其基本电生理学特性,同时建立BKCa动力学研究模型。结果构建稳定转染BKCa通道α和(或)β1亚单位的HEK293细胞系,通过膜片钳能够记录到具有与天然细胞BKCa电生理学特性相似的电流,包括大电导特性[内面向外式构型下单通道电导为(216.38±3.55)pS]、电压依赖性、Ca2+依赖性、钾离子选择性和200nmol/L IbTX(BKCa特异性阻断剂)阻断;联合表达β1亚单位能够明显增加通道的开放时程。采用inside-out宏观电流记录方式可以记录到明显的尾电流。可以用于分析通道激活、去激活等动力学模型及药物动力学作用机制。结论成功构建稳定转染BKCa通道α和(或)β1亚单位的HEK293细胞系。膜片钳实验证明具有典型的BKCa电生理学特性,同时建立的细胞系可以很好的用于BKCa通道功能调控研究和动力学参数分析。

Objective To construct the HEK 293 cell lines with stably expressed large-conductance calcium-activated potassium channels （BKca） and to investigate the basic characteristics and the kinetic model by using patch clamp technique. Methods pcD NA3.1-BKca and/or plRES-BKβ1 were transfected in HEK 293 cells with lipofectamine 2000 and the cell lines with stable expres- sion were constructed. The basic electrophysiological characteristics were investigated by patch clamp technique including single channels recording and whole cell recording and the kinetic model of BKca was constructed in inside-out and outside-out configura- tion. Results The basic property of ceil lines with stably expressed BKca α With/without β1 subunits was consistent with that in n- ative cells, including large conductance （216.38 ± 3.55 pS in insideout patches）, voltage dependence, calcium dependence, K＋ ion se- lectivity and being stocked by IbTX, a special blocker of BKca. BKca macro-currents and STOCs were significantly suppressed by 200 nmol/L IbTX,a specific blocker of BKca. β1 subunits increased the open duration. The tail currents were recorded by macro-in- sideout configuration. These currents could be used for analyzing the kinetics including the activation and deactivation and the mech- anism of drugs. Conclusion The HEK 293 cell lines with stably expressed BKca α and/or β1 are constructed successfully,which has typical electrophysiologieal properties of BKca. The cell lines can be used for studying the kinetic modulation of BKca