Human cell mutants affected in the interaction of the 12 beta-OH group of cardiac glycosides with the digitalis receptor.
The cross-resistance patterns of two different types of mutants of HeLa cells selected for resistance to the digoxin analog SC4453 (SCR mutants) in which the Na+/K+-ATPase is affected [A. Chopra and R. S. Gupta, J. biol. Chem. 261, 2034 (1986)], and towards numerous cardiac glycosides (CGs) and genins, were examined. One type of SCR mutant (designated as group C) was highly resistant to all CGs and genins investigated. In contrast, the other type of SCR mutant (group D) showed a high degree of cross-resistance towards selected CG derivatives (viz. digoxin, SC4453, digoxigenin, lanatoside C, alpha- and beta-methyldigoxin, dihydrodigoxin, alpha- and beta-acetyldigoxin, alpha,beta-diacetyldigoxin), all of which contained a free 12 beta-OH group in the steroid structure. Slight cross-resistance of the group D mutants was also observed for other compounds (viz. ouabain, ouabagenin, dihydroouabain) that contain a free 11 alpha-OH group in the molecule. However, these mutants exhibited no cross-resistance to other CG derivatives, which either lacked the above groups (viz. digitoxin, digitoxigenin, dihydrodigitoxin, digitoxigenin mono- and bisdigitoside, nerifolin, gitoxigenin, gitoxin, 16-acetylgitoxin, lanatosides A and B, cymarin, convallatoxin, oleandrin, strophanthidin, actodigin and bufalin) or in which the 12 beta-OH group was acetylated (viz. as in the case of 12-acetyldigoxin). Since the 12 beta-OH group is not required for CG-like activity, to account for these observations it is suggested that the genetic lesion in the group D mutant leads to the creation of a new binding site in the digitalis receptor, which specifically interacts with the 12 beta-OH group (the site presumably also interacts weakly with the 11 alpha-OH group) and either prevents or distorts the binding of the compounds to the drug binding site on the receptor. Further investigations with the different classes of CG-resistant mutants at the molecular level should prove very useful in identifying the drug receptor site and in understanding how these drugs interact with it.