The physiological significance of the cardiotonic steroid/ouabain-binding site of the Na,K-ATPase

Abstract

The Na,K-ATPase is the membrane "pump" that generates the Na(+) and K(+) gradients across the plasma membrane that drives many physiological processes. This enzyme is highly sensitive to inhibition by cardiotonic steroids, most notably the digitalis/ouabain class of compounds, which have been used for centuries to treat congestive heart failure and arrhythmias. The amino acids that constitute the ouabain-binding site are highly conserved across the evolutionary spectrum. This could be fortuitous or could result from this site being conserved because it has an important biological function. New physiological approaches using genetically engineered mice are being used to define the biological significance of the "receptor function" of the Na,K-ATPase and its regulation by potential endogenous cardiotonic steroid-like compounds. These studies extend the reach of earlier studies involving the biochemical purification of endogenous regulatory ligands.

Figure 1

Transmembrane and extracellular amino acid sequence of the α1 and α2 isoforms of the mouse Na,K-ATPase. The amino acids responsible for the differential sensitivity of the α1 and α2 isoforms are shown in red. The substitutions introduced into the mouse α1 and α2 isoforms that change their sensitivity to ouabain are shown in black. (a) The amino acid sequence of the α1 isoform and amino acid substitutions that convert this relatively ouabain-resistant isoform to one that is sensitive. The amino acids introduced are those occurring in the ouabain-sensitive human α1 isoform. (b) The mouse α2 isoform and the amino acid substitutions that convert this ouabain-sensitive isoform to one that is relatively ouabain resistant.

Figure 2

ACTH-induced hypertension is dependent on the ouabain-binding site of α2 isoform of the Na,K-ATPase. Systolic blood pressure (SBP) was measured in wild-type mice α1^R/Rα2^S/S and genetically engineered mice with an ouabain-resistant α2 isoform, α1^R/Rα2^R/R, before and during treatment with ACTH. Mice with either of these two genotypes were also injected with saline to serve as controls. Although animals with an α1^R/Rα2^S/S genotype exhibited an increase in blood pressure following administration of ACTH, those with the α1^R/Rα2^R/R genotype failed to increase blood pressure following ACTH administration. The blood pressure of mice with either of the two genotypes did not increase with saline administration. These studies clearly demonstrate that the ouabain-binding site of the α2 isoform of the Na,K-ATPase is required for ACTH-induced hypertension in mice. This figure is modified from figure 1a of Reference .

Figure 3

(a) Pharmacological activities of cardiotonic steroids such as ouabain. Pharmacological levels of ouabain inhibit the Na,K-ATPase to such an extent that intracellular Na⁺ increases, which, in turn, raises intracellular Ca²⁺ through the Na⁺/Ca²⁺ exchanger. The increase in Ca²⁺ then increases vascular muscle contraction. Physiological levels of endogenous ligands, which raise the concentration of intracellular Na⁺ and thus Ca²⁺, would be expected to act similarly. These concentrations of ouabain would also activate Na,K-ATPase-mediated signaling pathways. (b) Stress, volume, pregnancy. The effect of physiological levels of endogenous ouabain or ouabain-like compounds. With physiological levels of endogenous ligand such as endogenous ouabain or marinobufagenin, only the signaling pathway may be activated, as only a few molecules of Na,K-ATPase are needed to bind to the ligand as signaling responses are amplified. As only a few molecules of Na,K-ATPase are inhibited, this binding is unlikely to raise intracellular Na⁺ levels sufficiently to increase intracellular Ca²⁺ and activate vascular contraction. Still possible is that local concentrations of Na⁺ increase sufficiently with low levels of endogenous Na,K-ATPase ligands to alter vascular tone, as described (–122).