Expression Of The Human HCN2 Pacemaker Channel Transfected Into Mammalian Cells With Lipofectin

Objective Plasmid hHCN2/pcDNA3 was transfected into mammalian cells, including human embryonic kidney epithelial cells (HEK293) and neonatal rat cardiomyocytes. We studied the electrophysiological properties of the hHCN2 channel in the stably transfected-hHCN2 HEK293 cells and detected acute effects of amiodarone on cloned hHCN2 channel. We also studied the pacemaker current and the expression of HCN mRNA in neonatal rat cardiomyocytes. the spontenous beat change after plasmid hHCN2/pcDNA3 transfected into the neonatal rat cardiomyocytes and discussed whether the native pacemaker current is essential for cardiac pacing.Methods The plasmid hHCN2/pcDNA3 was transformed into E. Coli JM109 by calcium phosphate precipitation technique. The bacterial plasmid was prepared by using alkali lysis buffer and confirmed by restriction enzymes digestion.The plasmid was extracted in mid amount with the Genopure Plasmid Midi Kit and transfected into HEK293 cell lines with Lipofectin. The transfected cells would be survived in the medium containing G418 antibiotic. In conventional whole cell patch clamp experiments using HEK293-hHCN2 cells, HCN2 current (IhHCN2) was recorded. To observe the acute effects of amiodarone , cells were exposed acutely to different concentration of amiodarone.The expression levels of hHCN2 mRNA and protein were detected by RT-PCR and Western blot, respectively. The Plasmid hHCN2/pcDNA3 was also transiently transfected into the neonatal rat cardiomyocytes with Lipofectin. RT-PCR and Western blot were also used to detect the expression of the mRNA and protein of hHCN2 channel. The beats of neonatal rat cardiomyocytes were recorded.Results:1. The G418 resistant(600ug/ml) HEK293 cell lines were successfully established. Whole-cell patch clamp recorded a Cs+-sensitive inward current from–40mV to–140mV in response to hyperpolarization in transiently and stably transfected HEK293 cells. IhHCN2...