Selectivity of digitalis glycosides for isoforms of human Na,K-ATPase

Abstract

There are four isoforms of the alpha subunit (alpha1-4) and three isoforms of the beta subunit (beta1-3) of Na,K-ATPase, with distinct tissue-specific distribution and physiological functions. alpha2 is thought to play a key role in cardiac and smooth muscle contraction and be an important target of cardiac glycosides. An alpha2-selective cardiac glycoside could provide important insights into physiological and pharmacological properties of alpha2. The isoform selectivity of a large number of cardiac glycosides has been assessed utilizing alpha1beta1, alpha2beta1, and alpha3beta1 isoforms of human Na,K-ATPase expressed in Pichia pastoris and the purified detergent-soluble isoform proteins. Binding affinities of the digitalis glycosides, digoxin, beta-methyl digoxin, and digitoxin show moderate but highly significant selectivity (up to 4-fold) for alpha2/alpha3 over alpha1 (K(D) alpha1 > alpha2 = alpha3). By contrast, ouabain shows moderate selectivity ( approximately 2.5-fold) for alpha1 over alpha2 (K(D) alpha1 <or= alpha3 < alpha2). Binding affinities for the three isoforms of digoxigenin, digitoxigenin, and all other aglycones tested are indistinguishable (K(D) alpha1 = alpha3 = alpha2), showing that the sugar determines isoform selectivity. Selectivity patterns for inhibition of Na,K-ATPase activity of the purified isoform proteins are consistent with binding selectivities, modified somewhat by different affinities of K(+) ions for antagonizing cardiac glycoside binding on the three isoforms. The mechanistic insight on the role of the sugars is strongly supported by a recent structure of Na,K-ATPase with bound ouabain, which implies that aglycones of cardiac glycosides cannot discriminate between isoforms. In conclusion, several digitalis glycosides, but not ouabain, are moderately alpha2-selective. This supports a major role of alpha2 in cardiac contraction and cardiotonic effects of digitalis glycosides.

FIGURE 1.

Purification of human α1β1, α2β1, and α3β1 isoform complexes. Coomassie-stained Laemmli SDS-PAGE of purified isoform complexes at 5 μg of protein per lane is shown.

FIGURE 2.

Competitive displacement of [³H]ouabain by cardiac glycosides and aglycones. Representative experiments for displacement of [³H]ouabain by digoxin (upper left), digitoxin (upper middle), β-methyl digoxin (upper right), digoxigenin (lower left), digitoxigenin (lower middle), and bufalin (lower right) are shown. ●, α1; ▴, α2; ■, α3. The concentration of [³H]ouabain was 1 nm. Solid lines are the fitted curves for a one-site competitive displacement model (see “Experimental Procedures”).

FIGURE 3.

Dependence of isoform selectivity on the number of digitoxose residues. The ratios of K_D ±S.E. for α1/α2 and α1/α3 are taken from Table 1 (Group B).

FIGURE 4.

[³H]Digoxin-K⁺ antagonism (A) and [³H]digoxin dissociation rates (B). ●, α1; ▴, α2; ■, α3. A, [³H]digoxin binding in the presence of 0–10 mm K⁺ is shown. Average K_0.5K values based on single site inhibition for two separate experiments are shown. B, digoxin 1 mm was added to the membranes pre-bound with [³H]digoxin, and the remaining bound [³H]digoxin was measured at 37 °C at the indicated times.

FIGURE 5.

Inhibition of Na,K-ATPase activity of purified isoform complexes. Shown are representative experiments for inhibition of Na,K-ATPase activity by digoxin (upper left), digoxigenin (upper right), and β-methyl digoxin (lower left, 5 mm K⁺; lower right, 20 mm K⁺). ●, α1; ▴, α2■, α3. Solid lines are the fitted curves for a one-site inhibition model (see “Experimental Procedures”).

FIGURE 6.

Structure of Na,K-ATPase with bound ouabain. The figure shows the Protein Data Bank codes 3A3Y structure (38). Residues identical in human α2 and α3 and different in α1 are shown in cyan. Residues and numbering are those for human α1. Dark green, ouabain, steroid-lactone; light green, rhamnose. The figure was drawn by PYMOL.