Different combinations of insect Na,K-ATPase α- and β-subunit paralogs enable fine tuning of toxin resistance and enzyme kinetics

BackgroundCardiac glycosides are known to fatally inhibit the Na,K-ATPase throughout the animal kingdom. Several animals, however, evolved target-site insensitivity by substitutions in the otherwise highly conserved cardiac glycoside binding pocket located on the Na,K-ATPase -subunit. The minimal functional enzyme consist of an - and a {beta}-subunit, the latter considered mainly as a chaperone responsible for correct folding and membrane integration. We here analyze resistance to cardiac glycosides and kinetic properties of different Na,K-ATPase /{beta}-combinations of the large milkweed bug, Oncopeltus fasciatus. These insects have adapted to high concentrations of cardiac glycosides in their food plants via several rounds of Na,K-ATPase gene duplications followed by differential resistance conferring substitutions and subfunctionalization of the enzymes. ResultsTo investigate their characteristics we expressed nine combinations of O. fasciatus Na,K-ATPase /{beta}-sunbunits (three each) in Sf9 cells and tested them with two structurally distinct cardiac glycosides, calotropin, a host plant compound, and ouabain, a commonly used toxin. Differences in the number and identity of amino acid substitutions in the cardiac glycoside binding site resulted in large differences in activity and toxin resistance of the three -subunits. The enzymes kinetics were also influenced by the {beta}-subunits leading to increased activities (C{beta}3) or altered resistances. The host plant toxin calotropin proved to be a much more potent inhibitor than ouabain for the phylogenetically oldest C based enzymes. This effect was compensated for in the B and A based enzymes with A{beta}1 having higher resistance against calotropin than against ouabain. ConclusionThe originally higher inhibitory potency of the host compound calotropin supports a coevolutionary escalation of plant defenses and herbivore tolerance mechanisms. For the bugs the possession of multiple paralogs improved adaptation to plant toxins in a stepwise manner and mitigates pleiotropic effects by a compromise between ion pumping activity and resistance.