Amino acid substitutions of Na,K-ATPase conferring decreased sensitivity to cardenolides in insects compared to mammals

作者：Dalla Safaa; Swarts Herman G P; Koenderink Jan B; Dobler Susanne^*

来源：Insect Biochemistry and Molecular Biology, 2013, 43(12): 1109-1115.

DOI：10.1016/j.ibmb.2013.09.006

• Na,K-ATPase
• Cardenolide-adapted insects
• Decreased sensitivity to ouabain
• Site directed mutagenesis
• Baculovirus expression
• Sf9 cells

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 alpha-subunit of Drosophila melanogaster together with the beta-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 mu M) or T797A (63.3 mu M) was increased roughly 250-fold relative to the wild-type (0.24 mu M), the double mutations of Q111V-N122H (IC50 550 mu M) and Q111T-N122H (IC50 583 mu M) 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.

• 出版日期2013-12