Substitutions in the cardenolide binding site and interaction of subunits affect kinetics besides cardenolide sensitivity of insect Na,K-ATPase

Substitutions within the cardenolide target site of several insects' Na,K-ATPase α-subunits may confer resistance against toxic cardenolides. However, to which extent these substitutions alter the Na,K-ATPase's kinetic properties and how they interact with different β-subunits is not clear. The cardenolide-adapted milkweed bug Oncopeltus fasciatus possesses three paralogs of the α-subunit (A, B, and C) that differ in number and identity of resistance-conferring substitutions. We introduced these substitutions into the α-subunit of Drosophila melanogaster and combined them with the β-subunits Nrv2.2 and Nrv3. The substitutions Q111T-N122H-F786N-T797A (A-copy mimic) and Q111T-N122H-F786N (B-copy mimic) mediated high insensitivity to ouabain, yet they drastically lowered ATPase activity. Remarkably, the identity of the β-subunit was decisive and all α-subunits were less active when combined with Nrv3 than when combined with Nrv2.2. Both the substitutions and the co-expressed β-subunit stro...