COMPARISON OF OUABAIN-SENSITIVE AND OUABAIN-INSENSITIVE NA K PUMPS INHEK293 CELLS/

The Na/K pump current I-p of single HEK293 cells either untransfected (endogenous I-p) or transfected with the al subunit of the rat Na/K pu mp (exogenous I-p) was investigated in Na-containing solution by means of whole-cell recording at 30 degrees C. The endogenous I-p was irrev ersibly blocked by 10(-4) M ouabain or 2.10(-4) M dihydro-ouabain (DHO ). Its density amounted to 0.33 pA pF(-1) at 0 mV and 5.4 mM K-o. It w as 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 alpha 1 subunit of the rat Na/K pump showed an I- p in the presence of 10(-4) M ouabain and 2.10(-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 am plitude increased linearly with depolarization at negative voltages bu t remained almost constant at positive membrane potentials. Comparison with the I-p of isolated rat cardiac ventricular myocytes strongly su ggests that the exogenous I-p in HEK293 cells is generated by the alph a 1 subunit of the rat Na/K pump since it displays identical propertie s. Therefore, HEK293 cells represent an expression system well suited for the electrophysiological analysis of recombinant, cardiac glycosid e-resistant Na/K pumps by means of whole-cell recording.