MEDIATION OF CARDIAC GLYCOSIDE INSENSITIVITY IN THE MONARCH BUTTERFLY(DANAUS-PLEXIPPUS) - ROLE OF AN AMINO-ACID SUBSTITUTION IN THE OUABAIN BINDING-SITE OF NA-ATPASE(,K+)

The Monarch butterfly (Danaus plexippus) sequesters cardiac glycosides (CG) for its chemical defense against predators. Larvae and adults of this butterfly are insensitive towards dietary cardiac glycosides, wh ereas other Lepidoptera are sensitive and intoxicated by ouabain. Ouab ain inhibits Na+,K+-ATPase by binding to its alpha-subunit. We have am plified and cloned the DNA-sequence encoding the respective ouabain bi nding site. Instead of the amino acid asparagine at position 122 in ou abain-sensitive insects, the Monarch has a histidine in the putative o uabain binding site, which consists of 12 amino acids. Starting with t he CG-sensitive Na+,K+-ATPase gene from Drosophila, we converted pos. 122 to a histidine residue as in Danaus plexippus by site-directed mut agenesis. Human embryonic kidney cells (HEK) (which are sensitive to o uabain) were transfected with the mutated Na+,K+-ATPase gene in a pSVD F-expression vector and showed a transient expression of the mutated D rosophila Na+,K+-ATPase. When treated with ouabain, the transfected ce lls tolerated ouabain at a concentration of 50 mM, whereas untransform ed controls or controls transfected with the unmutated Drosophila gene , showed a substantial mortality. This result implies that the asparag ine to histidine exchange contributes to ouabain insensitivity in the Monarch. In two other CG-sequestering insects, e.g., Danaus gilippus a nd Syntomeida epilais, the pattern of amino acid substitution differed , indicating that the Monarch has acquired this mutation independently during evolution.