Ouabain Binding Kinetics of the Rat Alpha Two and Alpha Three Isoforms of the Sodium-Potassium Adenosine Triphosphate

The Na,K-ATPase has three α isoforms which differ in cardiac glycoside sensitivity and tissue distribution. The rodent α1 isoform is relatively resistant to cardiac glycosides, while the α2 and α3 isoforms are quite sensitive. Because both the α2 and α3 isoforms are generally expressed in the same tissue, it has been difficult to differentiate and accurately determine the kinetics of ouabain binding to these isoforms. To more fully understand the interactions of the α2 and α3 isoforms with cardiac glycosides, the association and dissociation rates of ouabain binding were measured in transfected cell lines. cDNA′s coding for the rat α2 and α3 isoforms were transfected into NIH 3T3 cells and characterized by Na,K-ATPase activity and [3H]ouabain binding. By individually expressing the α2 and α3 isoforms in ouabain-insensitive NIH 3T3 cells, the ouabain-binding characteristics of each isoform could be accurately determined. The association rate constants of the α2 and α3 isoforms were similar while the dissociation rate constant was 33 times slower for the α3 isoform than the α2 isoform. Calculation of the dissociation constant (Kd) from these rate constants yielded values of 115 and 1.6 nM for rat α2 and α3 isoforms, respectively. Scatchard analysis of the rat α2 isoform produced a similar value for Kd of 37 ± 9 nM. Inhibition of Na,K-ATPase activity indicates the rodent α1 isoform has an IC50 1000-fold higher than the α2 or α3 isoform at 4.8 × 10−5 M. The results are consistent with the hypothesis that the order of ouabain affinity between the rat α isoforms of the Na,K-ATPase is α3 > α2 ⪢ α1.