起搏通道HCN2基因稳定转染HEK293细胞的电生理特点

Electrophysiological properties of the human HCN2 pacemaker channel in stably transfected HEK293 cell lines.

目的了解超极化激活环核苷酸门控阳离子通道基因亚型HCN2(hHCN2)稳定转染人胚肾细胞(HEK293)的离子通道电生理特点。方法采用全细胞膜片钳技术测定hHCN2稳定转染HEK293细胞的通道电流(IhHCN2)电生理特点。结果人全长起搏通道HCN2(hHCN2)基因稳定转染HEK293,记录到一系列超极化内向离子流,该电流激活缓慢,呈电压、时间依赖性,没有明显失活过程,激活电位、半最大激活电位分别为-87±8mV(P>0.05)、-98±2mV(n=20),激活时间常数为196±36ms。起搏电流阻断剂ZD7288和Cs+灌流,IhHCN2峰值幅度均降低,半效抑制浓度分别为23.9±5.9μmol/L、126.8±27.1μmol/L,ZD7288的作用冲洗后抑制作用不能恢复,Cs+的作用在冲洗后基本恢复。结论稳定转染HEK293细胞的克隆起搏通道hHCN基因具有起搏电流的特点。

Objective To study the electrophysiological properties of the human hyperpolarization activated cyclic nucleotide gate cation channel 2 （ hHCN2 ） stably transfected into human embryonic kidney epithelial cell （ HEK293 ）. Methods The electrophysiological properties of the hHCN2 channel current in the stable HEK293 cell lines transfected with pacemaker channel human HCN2 gene was recorded by whole-cell patch clamp . Results An inward current was recorded from -40 mV to - 140 mV in response to hyperpolarization in stably transfected HEK293 cells. The hHCN2 channel current （ IhHCN2 ） began to slowly activate in voltage and time dependent manner without deactivation. Threshold voltage （Vlh） and potential of half-maximal activation （ V1/2 ） was - 87 ± 8 mV, - 98 ± 2 mV, respectively. Time constants of channel activation was 196 ± 36 ms （at - 140 mV） by exponential function fitting the current. Two of If inhibitors, ZD7288 and Cs^＋ , decreased the peak current of IhHCN2 and their IC50 were 23.9 ± 5.9 μmoL/L, 126.8 ± 27.1 μmoL/L, respectively. ZD7288 block was not released by （extracellular） washout. The release of Cs^＋ block was equally rapid upon switching back to bath solution without Cs^＋. Conclusion When stably expressed in HEK293 cells, hHCN2 channel gives rise to inward cation current with the hallmark features of native If.