[Electrophysiology of two human hyperpolarization activated cyclic nucleotide-gated potassium channels expressed in HEK293 cells].
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 transfected 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. Boltzmann 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 both currents.


CONCLUSIONS
The target genes hHCN2 and hHCN4 are successfully expressed in HEK293 cells, and the expressed functional channels have profoundly different activation kinetics.
Li
Chun
C
Department of Cardiology, People's Hospital, Peking University, Beijing 100044, China.
Guo
Ji-Hong
JH
Li
Ji-Wen
JW
Liu
Yuan-Wei
YW
Hao
Xue-Mei
XM
Wang
Shi-Qiang
SQ
D001692:Biological Transport / Q000187:drug effects; D002460:Cell Line; D002586:Cesium / Q000494:pharmacology; D002712:Chlorides / Q000494:pharmacology; D054815:Cyclic Nucleotide-Gated Cation Channels / Q000235:genetics / Q000502:physiology*; D006801:Humans; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; D007473:Ion Channels / Q000235:genetics / Q000502:physiology*; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D009124:Muscle Proteins / Q000235:genetics / Q000502:physiology*; D018408:Patch-Clamp Techniques; D011188:Potassium / Q000378:metabolism; D015221:Potassium Channels; D012964:Sodium / Q000378:metabolism; D014162:Transfection
D002712:Chlorides; D054815:Cyclic Nucleotide-Gated Cation Channels; C578710:HCN2 protein, human; C119587:HCN4 protein, human; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; D007473:Ion Channels; D009124:Muscle Proteins; D015221:Potassium Channels; D002586:Cesium; D012964:Sodium; C028019:cesium chloride; D011188:Potassium