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, whereas other Lepidoptera are sensitive and intoxicated by ouabain. Ouabain inhibits Na(+),K(+)-ATPase by binding to its α-subunit. We have amplified and cloned the DNA-sequence encoding the respective ouabain binding site. Instead of the amino acid asparagine at position 122 in ouabain-sensitive insects, the Monarch has a histidine in the putative ouabain binding site, which consists of 12 amino acids. Starting with the CG-sensitive Na(+),K(+)-ATPase gene fromDrosophila, we converted pos. 122 to a histidine residue as inDanaus plexippus by site-directed mutagenesis. Human embryonic kidney cells (HEK) (which are sensitive to ouabain) were transfected with the mutated Na(+),K(+)-ATPase gene in a pSVDF-expression vector and showed a transient expression of the mutatedDrosophila Na(+),K(+)-ATPase. When treated with ouabain, the transfected cells tolerated ouabain at a concentration of 50 mM, whereas untransformed controls or controls transfected with the unmutatedDrosophila gene, showed a substantial mortality. This result implies that the asparagine to histidine exchange contributes to ouabain insensitivity in the Monarch. In two other CG-sequestering insects, e.g.,Danaus gilippus andSyntomeida epilais, the pattern of amino acid substitution differed, indicating that the Monarch has acquired this mutation independently during evolution.

The Monarch (Danaus plexippus) sequesters cardiac glycosides for its chemical defence against predators. Larvae and adults of this butterfly are insensitive towards dietary cardiac glycosides, whereas other Lepidoptera, such as Manduca sexta and Creatonotos transiens are sensitive and intoxicated by ouabain. Ouiabain inhibits the Na+,K+‐ATPase by binding to its α‐subunit. We have amplified and cloned the DNA sequence encoding the respective ouabain binding site. Instead of the amino acid asparagine at position 122 in ouabain‐sensitive insects, the Monarch has a histidine in the putative ouabain binding site, which consists of about 12 amino acids. This change may explain the ouabain insensitivity.

Zentrum fur Molekulare Biologie Universitat Heidelberg 1m Neuenheimer Feld 282 6900 Heidelberg, FRG Signal transmission at chemical synapses involves specific receptors that transduce neurotransmitter binding into alterations of membrane potential. Receptors containing integral ion channels mediate rapid (in the ~ msec range) transduction events, whereas receptors activating G-protein coupled channels operate at slower time scales (in the msec to sec range). At resting membrane potential, excitation is generated by cation influx, but inhibition of neuronal firing results from increased chloride permeability.The nicotinic acetylcholine receptor at the neuromuscular junction initiates muscle contraction. Due to its abundance in the electric organ of certain fish species, it is the best characterized ion channel protein known (reviewed in Changeux et al., 1984). The primary structures of its subunits have been determined in different species, and homologous cDNAs have been isolated from vertebrate and Drosophila brain. The major inhibitory neurotra1).smitters at central synapses, glycine and ~-aminobutyric acid (GABA), gate chloride channel-forming receptors of similar conductance properties (Bormann et al., 1987), but dis tinc t pharmacology . For example, the convulsive alkaloid strychnine antagonizes postsynaptic inhibition by glycine, the predominant inhibitory neurotransmitter in brain stem and spinal cord, whereas benzodiazepines and barbiturates modify inhibitory GABAA receptor responses in many regions of the central nervous system.