Beneficent and Maleficent Effects of Cations on Bufadienolide Binding to Na⁺,K⁺-ATPase

Kinetic properties and crystal structures of the Na⁺,K⁺-ATPase in complex with cardiotonic steroids (CTS) revealed significant differences between CTS subfamilies (Laursen et al.). Thus, we found beneficial effects of K⁺ on bufadienolide binding, which strongly contrasted with the well-known antagonism between K⁺ and cardenolides. In order to understand this peculiarity of bufalin interactions, we used docking and molecular dynamics simulations of the complexes involving Na⁺,K⁺-ATPase, bufadienolides (bufalin, cinobufagin), and ions (K⁺, Na⁺, Mg²⁺). The results revealed that bufadienolide binding is affected by (i) electrostatic attraction of the lactone ring by a cation and (ii) the ability of a cation to stabilize and "shape" the site constituted by transmembrane helices of the α-subunit (αM1-6). The latter effect was due to varying coordination patterns involving amino acid residues from helix bundles αM1-4 and αM5-10. Substituents on the steroid core of a bufadienolide add to and modify the cation effects. The above rationale is fully consistent with the ion effects on the kinetics of Na⁺,K⁺-ATPase/bufadienolide interactions.