Characterization of two classes of cardiac glycosine binding sites in rat heart and brain membrane preparations, using quantitative computer modelling.

Cardiac glycoside binding to rat heart and brain membrane preparations was measured by a rapid filtration technique. Data were analysed using quantitative computer analysis. The experimental results were consistent with a model in which cardiac glycoside-specific binding occurs at two independent classes of sites. The high-affinity sites in heart membranes were characterized by dissociation constants (Kd) of 40, 50, and 61 nmol/l for ouabain, digoxin and digitoxin, respectively, and the lower-affinity sites were characterized by Kd of 2.3 mumol/l, 67 nmol/l and 71 nmol/l for ouabain, digoxin and digitoxin, respectively. Comparable results were obtained using brain membranes. Potassium ions inhibit [3H]-ouabain binding in a dose-dependent manner with an IC50 of 500 mumol/l. Quantitative computer modeling indicated that potassium inhibits ouabain binding to approximately the same extent at both classed of binding sites, consistent with the hypothesis that the two classes of binding sites for cardiac glycosides might be associated with the Na+K+-ATPase.