Identification of endogenous outward currents in the human embryonic kidney (HEK 293) cell line.
Human embryonic kidney cells (HEK 293) are widely used as an expression system in studies of ion channels. However, their endogenous ionic currents remain largely unidentified. To characterize these currents, we performed patch clamp experiments on this expression system. In whole-cell voltage clamp mode, the HEK 293 cells showed mainly outward currents using physiological concentrations of Na+ and K+ and symmetric concentrations of Cl- (150 mM) across the plasma membranes. K+ currents contributed to a small portion of these outward currents, since a shift of the reversal potentials of only approximately 20 mV was seen with a change of extracellular K+ concentration from 3 to 150 mM. In contrast, the reversal potential shifted approximately 25 mV when extracellular Cl- was reduced to 50 mM, indicating that most of the outward currents are carried by Cl-. In inside-out patches, several distinct Cl- currents were identified. They were: (1) 350 pS Cl- current, which was voltage-activated and had a moderate outward rectification; (2) 240 pS Cl- current with a weak outward rectification; and (3) 55 pS Cl- current, which was voltage-activated, sensitive to DIDS, and showed a strong outward rectification. Activation of these Cl- currents did not require an elevation of free Ca2+ level in the cytosol. Besides these three currents, we observed two other Cl- currents with much smaller conductances (25 and 16 pS, respectively). Two different K+ currents were seen in the HEK 293 cells, with one of them (125 pS) showing inward rectification and the other (70 pS) outward rectification. Moreover, a 50 pS cation channel was recorded in these cells. The presence of a variety of ion channels in the HEK 293 cells suggests that a great precaution needs to be taken when this expression system is used in studies of several similar ion channels.
Department of Biology, Georgia State University, Atlanta 30303-4010, USA.
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G
Zhu
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Zhang
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Xu
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Jiang
D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D018118:Chloride Channels / Q000502:physiology; D002712:Chlorides / Q000378:metabolism; D004622:Embryo, Mammalian; D006801:Humans; D007473:Ion Channels / Q000502:physiology*; D016623:Ion Pumps / Q000502:physiology; D007668:Kidney / Q000166:cytology; D008564:Membrane Potentials / Q000502:physiology; D018408:Patch-Clamp Techniques; D011188:Potassium / Q000378:metabolism; D015221:Potassium Channels / Q000502:physiology; D012964:Sodium / Q000378:metabolism
D018118:Chloride Channels; D002712:Chlorides; D007473:Ion Channels; D016623:Ion Pumps; D015221:Potassium Channels; D012964:Sodium; D011188:Potassium; D002118:Calcium