Na+,K+-ATPase Isoform Selectivity for Digitalis-Like Compounds Is Determined by Two Amino Acids in the First Extracellular Loop

Digitalis-like compounds (DLCs) comprise a diverse group of molecules characterized by a cis-trans-cis ring-fused steroid core linked to a lactone. They have been used in the treatment of different medical problems including heart failure, where their inotropic effect on heart muscle is attributed to potent Na+,K+-ATPase inhibition. Their application as drugs, however, has declined in recent past years due to their small safety margin. Since human Na+,K+-ATPase is represented by four different isoforms expressed in a tissue-specific manner, one of the possibilities to improve the therapeutic index of DLCs is to exploit and amend their isoform selectivity. Here, we aimed to reveal the determinants of selectivity of the ubiquitously expressed alpha 1 isoform and the more restricted alpha 2 isoform toward several well-known DLCs and their hydrogenated forms. Using baculovirus to express various mutants of the alpha 2 isoform, we were able to link residues Met119 and Ser124 to differences in affinity between the alpha 1 and alpha 2 isoforms to ouabain, dihydro-ouabain, digoxin, and dihydro-digoxin. We speculate that the interactions between these amino acids and DLCs affect the initial binding of these DLCs. Also, we observed isoform selectivity for DLCs containing no sugar groups.