Data from: 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 strongly affected the enyzme's affinity for Na+ and K+. Na+ affinity was considerably higher for all enzymes expressed with nrv3 while expression with nrv2.2 increased K+ affinity. Our results provide the first evidence that resistance against cardenolides comes at the cost of significantly altered kinetic properties of the Na,K-ATPase. The β-subunit can strongly modulate these properties but cannot fully compensate for the effect of the substitutions.几种昆虫的钠钾ATP酶（Na,K-ATPase）α亚基上的强心甾类化合物（cardenolide）作用位点发生的氨基酸替换，或可使其获得对抗有毒强心甾类化合物的抗性。然而，此类替换在多大程度上会改变钠钾ATP酶的动力学特性，以及它们与不同β亚基（β-subunit）的相互作用机制，目前仍未明确。适应强心甾类化合物的乳草长蝽（Oncopeltus fasciatus）拥有三种α亚基旁系同源基因（paralogs），分别为A型、B型与C型，三者携带的抗性相关替换的数量与种类均存在差异。我们将这些替换引入黑腹果蝇（Drosophila melanogaster）的α亚基中，并将重组后的α亚基分别与β亚基Nrv2.2和Nrv3进行共表达。携带Q111T-N122H-F786N-T797A（A型模拟替换）以及Q111T-N122H-F786N（B型模拟替换）的重组酶，对哇巴因（ouabain）表现出高度不敏感性，但同时显著降低了ATP酶活性。值得注意的是，β亚基的类型起到了决定性调控作用：所有与Nrv3组合的α亚基，其酶活性均低于与Nrv2.2组合的对应组别。氨基酸替换与共表达的β亚基均会显著影响该酶对钠离子（Na+）和钾离子（K+）的亲和力。当酶与nrv3共表达时，对Na+的亲和力显著升高；而与nrv2.2共表达时，则提升了对K+的亲和力。本研究结果首次证实，针对强心甾类化合物的抗性，需以钠钾ATP酶动力学特性发生显著改变为代价。β亚基可强烈调控此类动力学特性，但无法完全补偿氨基酸替换带来的影响。