HEK293细胞上表达的人类超极化激活环核苷酸门控钾通道亚型2和亚型4的电生理

Electrophysiology of two human hyperpolarization activated cyclic nucleotide-gated potassium channels expressed in HEK293 cells

目的将人类超极化激活环核苷酸门控钾通道亚型2(hHCN2)和亚型4(hHCN4)的基因表达于 HEK293细胞系上,观察其表达的通道的电生理特点。方法包含目的基因 hHCN2或hHCN4的 cNDA 亚克隆到腺病毒穿梭载体 pAdTrack-CMV,穿梭载体和病毒骨架载体 pAdEasy-1在大肠杆菌重组,病毒经过包装和扩增后分别转染 HEK293细胞。利用全细胞膜片钳技术,记录分别转染了 hHCN2和 hHCN4的 HEK293细胞上的超极化激活钾电流(I_f)。结果转染 hHCN2和 hHCN4的HEK293细胞上产生超极化激活的内向电流。两种通道的半数最大激活电压分别为-114.8 mV±3.3 mV 和-125.9 mV±2.9 mV,反向曲线斜率分别为11.1 mV±1.2 mV 和13.7 mV±1.3 mV。两者的激活动力学明显不同,刺激电压为-110 mV 时,通道的激活时间常数分别为0.99 s±0.21 s 和8.47 s±2.85 s。两种通道对钠离子和钾离子的相对通透性分别为0.40和0.34。两种电流均可被2mmol/L 的氯化铯(CsCl)阻断。结论目的基因 hHCN2和 hHCN4在 HEK293细胞上成功表达,形成的两种通道具有明显不同的激活动力学。

Objective To express the human HCN2 and HCN4 genes in HEK293 cells and investigate the electrophysiology of the expressed channel protein. Methods cDNA encoding human HCN2 or HCN4 gene was ligated into a shuttle vector pAdTrack-CMV. Homologous recombination was performed in Escherichia coli of the line BJ5183. Human embryonic kidney cells of the line 293 （ HEK293 cells） were cultured and transfectd with the positive recombinant adenovirus plasmid. Then the HEK293 cells were infected by AdhHCN2 or AdhHCN4 and the whole cell hyperpolarization-activated currents were recorded in HEK293 cells transfected with hHCN2 and hHCN4. Results If-like currents could be found in the HEK293 cells transfected with hHCN2 and hHCN4. The channels were activated by hyperpolarized potentials. Boltamann equation showed that the half-activation voltage of the hHCN2 and hHCN4 channels were - 114. 8 mV ±3.3 mV and -125.9 mV ± 2.9 mV respectively （P = 0. 024）. The reversal slope factors of the hHCN2 and hHCN4 channels were 11.1 mV ± 1.2 mV and 13.7 mV ± 1.3 mV respectively （ P - 0.22）. The activation kinetics was faster in hHCN2 than in hHCN4, with the activation constants at - 110 mV being 0.99 s±0.21 s and 8.47 s ±2.85 s respectively. The relative permeation ratio for sodium and potassium were 0.40 and 0.34 respectively in these two channels. Caesium chloride of the concentration of 2 mmol/L prominently inhibited beth currents. Conclusion The target genes hHCN2 and hHCN4 are successfully expressed in HEK293 cells, and the expressed functional channels have profoundly different activation kinetics.