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.
Mogul
D J
DJ
Department of Electrical Engineering and Computer Science, Northwestern University, Chicago, IL 60611.
Rasmussen
H H
HH
Singer
D H
DH
Ten Eick
R E
RE
D000818:Animals; D001665:Binding Sites; D005260:Female; D006168:Guinea Pigs; D006352:Heart Ventricles; D066298:In Vitro Techniques; D008297:Male; D008954:Models, Biological; D009206:Myocardium / Q000166:cytology / Q000378:metabolism*; D010042:Ouabain / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D011188:Potassium / Q000378:metabolism*; D012964:Sodium / Q000378:metabolism*; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*
C000977:dihydroouabain; D010042:Ouabain; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; D011188:Potassium