Comparison of ouabain-sensitive and -insensitive Na/K pumps in HEK293 cells.
The Na/K pump current I(p) of single HEK293 cells either untransfected (endogenous I(p)) or transfected with the alpha1 subunit of the rat Na/K pump (exogenous I(p)) was investigated in Na-containing solution by means of whole-cell recording at 30 degrees C. The endogenous I(p) was irreversibly blocked by 10(-4) M ouabain or 2 x 10(-4) M dihydro-ouabain (DHO). Its density amounted to 0.33 pA pF(-1) at 0 mV and 5.4 mM K(o). It was half maximally activated at 1.5 mM K(o) and increased linearly with depolarization over the entire voltage range studied (-80 to +60 mV). In contrast, HEK293 cells stably transfected with cDNA for the cardiac glycoside-resistant alpha1 subunit of the rat Na/K pump showed an I(p) in the presence of 10(-4) M ouabain and 2 x 10(-4) M DHO, respectively. This exogenous I(p) was reversibly blocked by 10(-2) M ouabain. Half maximal activation of the exogenous I(p) occurred at 1.7 mM K(o). Its amplitude increased linearly with depolarization at negative voltages but remained almost constant at positive membrane potentials. Comparison with the I(p) of isolated rat cardiac ventricular myocytes strongly suggests that the exogenous I(p) in HEK293 cells is generated by the alpha1 subunit of the rat Na/K pump since it displays identical properties. Therefore, HEK293 cells represent an expression system well suited for the electrophysiological analysis of recombinant, cardiac glycoside-resistant Na/K pumps by means of whole-cell recording.
Kockskämper
J
J
Arbeitsgruppe Muskelphysiologie, Ruhr-Universität, Bochum, Germany.
Gisselmann
G
G
Glitsch
H G
HG
D000818:Animals; D002460:Cell Line / Q000187:drug effects / Q000201:enzymology; D004553:Electric Conductivity; D006801:Humans; D009206:Myocardium / Q000378:metabolism; D010042:Ouabain / Q000494:pharmacology*; D018408:Patch-Clamp Techniques; D011188:Potassium / Q000494:pharmacology; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D011994:Recombinant Proteins; D010042:Ouabain; D000254:Sodium-Potassium-Exchanging ATPase; D011188:Potassium