All human Na⁺-K⁺-ATPase α-subunit isoforms have a similar affinity for cardiac glycosides

Abstract

Three alpha-subunit isoforms of the sodium pump, which is the receptor for cardiac glycosides, are expressed in human heart. The aim of this study was to determine whether these isoforms have distinct affinities for the cardiac glycoside ouabain. Equilibrium ouabain binding to membranes from a panel of different human tissues and cell lines derived from human tissues was compared by an F statistic to determine whether a single population of binding sites or two populations of sites with different affinities would better fit the data. For all tissues, the single-site model fit the data as well as the two-site model. The mean equilibrium dissociation constant (K(d)) for all samples calculated using the single-site model was 18 +/- 6 nM (mean +/- SD). No difference in K(d) was found between nonfailing and failing human heart samples, although the maximum number of binding sites in failing heart was only approximately 50% of the number of sites in nonfailing heart. Measurement of association rate constants and dissociation rate constants confirmed that the binding affinities of the different human alpha-isoforms are similar to each other, although calculated K(d) values were lower than those determined by equilibrium binding. These results indicate both that the affinity of all human alpha-subunit isoforms for ouabain is similar and that the increased sensitivity of failing human heart to cardiac glycosides is probably due to a reduction in the number of pumps in the heart rather than to a selective inhibition of a subset of pumps with different affinities for the drugs.

Ouabain Binds with High Affinity to the Na,K-ATPase in Human Polycystic Kidney Cells and Induces Extracellular Signal–Regulated Kinase Activation and Cell Proliferation

“…Normal kidney expresses predominantly one isozyme of the Na,KATPase, composed of ␣1 and ␤1 isoforms (3,4). In our study, the K i for the more resistant Na,K-ATPase component that was observed in both the NHK and ADPKD cells is consistent with the ouabain kinetics that correspond to the ␣1␤1 isozyme (37). The high ouabain-sensitive Na,K-ATPase that is unique to the cystic cells suggested the presence of additional ␣ or ␤ isoforms.…”

“…For this, dose-response curves for the inhibition of Na,K-ATPase activity by ouabain were determined ( Figure 7). NHK cells exhibited a monophasic ouabain inhibition profile ( Figure 7A), with a ouabain K i of 0.48 Ϯ 0.05 ϫ 10 Ϫ6 M, consistent with what has been described in normal kidney (3,4,37). In contrast, ADPKD cells showed a heterogeneous response, with two Na,K-ATPase populations with different affinities for ouabain.…”

The highly conserved cardiac glycoside binding site of Na,K-ATPase plays a role in blood pressure regulation

“…In humans, unlike rodents, the Na,K-ATPase ␣1 isoform is sensitive to cardiac glycosides (28,29). Thus, the cardiac glycoside binding site of the ␣1 isoform may play a biological role in hypertension similar to that of the ␣2 isoform.…”

“…Because the ␣1 isoform is ouabainresistant in mice, it is unlikely that it is a receptor for such ligands. However, in humans the ␣1 isoform is sensitive to cardiac glycosides and may be responsive to a naturally occurring ligand (28,29).…”

The α1 Isoform of Na,K-ATPase Regulates Cardiac Contractility and Functionally Interacts and Co-localizes with the Na/Ca Exchanger in Heart

“…It is quite clear that inhibition of either the ␣1 or ␣2 isoform enhances cardiac contraction. This is of importance as human hearts express essentially equal amounts of three ␣ isoforms (␣1, ␣2, and ␣3), which are all sensitive to ouabain (10,28). Thus, in patients with congestive heart failure, inhibition of the ␣1 isoform along with the ␣2 isoform by cardiac glycosides will contribute to increased heart contraction.…”