title: Amino acid substitutions of Na,K-ATPase conferring decreased sensitivity to cardenolides in insects compared to mammals.
abstract: Mutagenesis analyses and a recent crystal structure of the mammalian Na,K-ATPase have identified amino acids which are responsible for high affinity binding of cardenolides (such as ouabain) which at higher doses block the enzyme in the phosphorylated state. Genetic analysis of the Na,K-ATPase of insects adapted to cardenolides in their food plants revealed that some species possess substitutions which confer strongly increased resistance to ouabain in the mammalian enzyme such as the substitution T797A or combined substitutions at positions 111 and 122. To test for the effect of these mutations against the background of insect Na,K-ATPase, we here expressed the ouabain sensitive Na,K-ATPase α-subunit of Drosophila melanogaster together with the β-subunit Nrv3 in baculovirus-infected Sf9 cells and introduced the substitutions N122H, T797A, Q111T-N122H, Q111V-N122H, all of which have been observed in cardenolide-adapted insects. While all constructs showed similar expression levels, ouabain affinity of mutated Na,K-ATPases was reduced compared to the wild-type fly enzyme. Ouabain sensitivity of the ATPase activity in inhibition assays was significantly decreased by all mutations, yet whereas the IC₅₀ for the single mutations of N122H (61.0 μM) or T797A (63.3 μM) was increased roughly 250-fold relative to the wild-type (0.24 μM), the double mutations of Q111V-N122H (IC₅₀ 550 μM) and Q111T-N122H (IC₅₀ 583 μM) proved to be still more effective yielding a 2.250-fold increased resistance to ouabain. The double mutations identified in cardenolide-adapted insects are more effective in reducing ouabain sensitivity of the enzyme than those found naturally in the rat Na,K-ATPase (Q111R-N122D) or in mutagenesis screens of the mammalian enzyme. Obviously, the intense selection pressure on cardenolide exposed insects has resulted in very efficient substitutions that decrease cardenolide sensitivity extremely.
authors.affiliation: Molecular Evolutionary Biology, Biocenter Grindel, Universität Hamburg, Martin-Luther-King Pl. 3, 20146 Hamburg, Germany.
authors.forename: Safaa
authors.initials: S
authors.lastname: Dalla
authors.forename: Herman G P
authors.initials: HG
authors.lastname: Swarts
authors.forename: Jan B
authors.initials: JB
authors.lastname: Koenderink
authors.forename: Susanne
authors.initials: S
authors.lastname: Dobler
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics*; D000818:Animals; D002298:Cardenolides / Q000494:pharmacology*; D029721:Drosophila Proteins / Q000235:genetics / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000201:enzymology; D017136:Ion Transport / Q000235:genetics; D016296:Mutagenesis; D009154:Mutation / Q000187:drug effects; D010042:Ouabain / Q000494:pharmacology; D051381:Rats; D000254:Sodium-Potassium-Exchanging ATPase / Q000235:genetics / Q000378:metabolism*
chemical_list: D002298:Cardenolides; D029721:Drosophila Proteins; D010042:Ouabain; C472021:ATPalpha protein, Drosophila; D000254:Sodium-Potassium-Exchanging ATPase
keywords: Baculovirus expression; Cardenolide-adapted insects; Decreased sensitivity to ouabain; Na,K-ATPase; Sf9 cells; Site directed mutagenesis