NA PUMP CURRENT CAN BE SEPARATED INTO OUABAIN-SENSITIVE AND OUABAIN-INSENSITIVE COMPONENTS IN SINGLE-RAT VENTRICULAR MYOCYTES

The present study was undertaken to identify functional isoforms of th e Na,K-ATPase in single rat cardiac myocytes, Na,K-ATPase activity was measured as ouabain-sensitive, extracellular K-activated outward curr ent (Na pump current) in ventricular myocytes voltage-clamped with sin gle low-resistance (0.5-1 M Omega) patch electrodes at 36 degrees C. S olutions to block contaminating currents allowed Na pump current to be measured without significant contamination in 140 mM Na-containing su perfusion solutions, The current-voltage relationship had a positive s lope at potentials from -125 to 0 mV but became almost voltage-indepen dent at positive potentials, The apparent K-m for activation of this c urrent at -40 mV by extracellular K was 2.7+/-0.3 mM (mean+/-SEM, n=3) and increasing electrode Na increased the amplitude of the current to a maximum density of 4.11+/-0.17 pA/pF (n=34). Intracellular vanadate (100 mu M) produced an extracellular K-dependent inhibition of Na pum p current that was rapidly reversed in K-free superfusion solution, Do se-dependent inhibition of Na pump current by ouabain was best describ ed as the sum of two Michaelis-Menten binding sites: one with higher a ffinity (K-1/2=1.0+/- 0.7 mu M) comprising 33+/-9% (n=5-6) of the tota l current and the second with a K-1/2 of 43+/-14 mu M. Changing electr ode [Na] from 15 to 100 mM had no effect on the dose-dependent inhibit ion of the current by ouabain, Thus, the properties of high and low af finity components of Na pump current are consistent with the presence of different Na,K-ATPases isoforms that have a similar ion dependence for transport activity.