Endogenous channels in HEK cells and potential roles in HCN ionic current measurements.
A transformed line of human embryonic kidney epithelial cells (HEK 293) is commonly used as an expression system for exogenous ion channel genes. Previously, it has been shown that these cells contain mRNAs for a variety of ion channels. Expression of some of these genes has been confirmed at the protein level. Patch-clamp electrophysiology experiments confirm the presence of multiple ion channels and molecular data agree with pharmacological profiles of identified channels. In this work, we show that endogenous voltage-gated potassium channels in HEK cells are a significant source of outward current at positive potentials. We show that both non-transfected HEK cells and HEK cells transfected with hyperpolarization-activated cyclic-nucleotide gated (HCN) channels have a significant amount of voltage-gated potassium (K(V)) current when certain tail current voltage-clamp protocols are used to assay HCN current activation. Specifically, tail current protocols that use a depolarized holding potential of -40 mV followed by hyperpolarizing pulses (-80 to -140 mV) and then a tail pulse potential of +20 mV indicate K(V) channels undergo closed-state inactivation at the more depolarized holding potential of -40 mV, followed by recovery from inactivation (but no activation) at hyperpolarizing potentials and high amount of activation at the positive tail potential. Our results indicate that pulse protocols with positive tail pulses are inaccurate assays for HCN current in certain HEK cells. Surprisingly, HEK-293 cells were found to contain mRNA for HCN2 and HCN3 although we have not detected a significant and consistent endogenous I(f)-like current in these cells.
CS Department, University of Wisconsin, River Falls, WI 54022, USA. anthony.varghese@uwrf.edu
Anthony
A
Varghese
Erica M
EM
Tenbroek
James
J
Coles
Daniel C
DC
Sigg
D002460:Cell Line; D054815:Cyclic Nucleotide-Gated Cation Channels; D006801:Humans; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; D015640:Ion Channel Gating; D007473:Ion Channels / Q000502:physiology*; D009124:Muscle Proteins / Q000502:physiology; D018408:Patch-Clamp Techniques; D015221:Potassium Channels; D024642:Potassium Channels, Voltage-Gated / Q000502:physiology
D054815:Cyclic Nucleotide-Gated Cation Channels; C119587:HCN4 protein, human; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; D007473:Ion Channels; D009124:Muscle Proteins; D015221:Potassium Channels; D024642:Potassium Channels, Voltage-Gated