Inhibition of Na-K Pump Current in Guinea Pig Ventricular Myocytes by Dihydroouabain Occurs at High- and Low-affinity Sites

Binding of cardiac glycosides to the Na+,K+-dependent ATPase has been shown to occur at both high- and low-affinity sites. However, recent reports suggest that glycoside-induced inhibition of electrogenic Na-K pump current occurs with simple first-order binding kinetics at relatively low-affinity sites. This implies that high-affinity binding sites have little to do with Na-K pump inhibition during exposure to cardiac glycosides. To better understand the role of the high-affinity site, we investigated the concentration dependence of Ipump inhibition by dihydroouabain (DHO) in guinea pig ventricular myocytes through use of wide-pore patch pipettes to "fix" internal Na+ activity at approximately 30 mM and to voltage clamp at -40 mV (T = 34 degrees C). DHO was found to have no effect on membrane conductance at a holding potential of -40 mV. Holding current was monitored and the difference between steady-state holding current before and during external exposure to nine concentrations (range, 0.01-1,000 microM) of DHO was measured and normalized to cellular membrane capacitance. The concentration dependence of the inhibition of Na-K pump current was biphasic and well fitted to a two-binding site model with inhibitory KD values of 0.05 microM and 64.5 microM. This is consistent with previously reported 3H-ouabain binding studies in guinea pig myocardium. These findings indicate that the electrogenic properties of the Na-K pump can be inhibited by glycoside binding to both high- and low-affinity sites.