DOI: 10.5061/dryad.j0zpc86dg
title: Different combinations of insect Na,K-ATPase α- and β-subunits enable fine-tuned adaptation to host plant toxins and tissue specific needs

description: Background Cardiac 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
 α-subunit. The minimal functional enzyme consist of an α- and a β-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
 α/β-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. Results To investigate their
 characteristics we expressed nine combinations of O. fasciatus Na,K-ATPase
 α/β-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 α-subunits. The enzymes’ kinetics were also influenced by the
 β-subunits leading to increased activities (αCβ3) or altered resistances.
 The host plant toxin calotropin proved to be a much more potent inhibitor
 than ouabain for the phylogenetically oldest αC based enzymes. This effect
 was compensated for in the αB and αA based enzymes with αAβ1 having higher
 resistance against calotropin than against ouabain. Conclusion The
 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.
description: Nine different Na,K-ATPase α/β-subunit combinations of
 <i>O. fasciatus </i>were expressed heterologously in Sf9 cells
 using the baculovirus expression system (Dalla & Dobler 2016,
 Evolution 70:2767–77)<i>.</i> Enzyme activities were
 determined by photometric measurements of inorganic phosphate released
 from enzymatic ATP hydrolysis while subtracting background ATPase activity
 following previously described methods (Dalla et al. 2017, Insect Biochem
 Mol Biol 89:43–50; Petschenka et al. 2013, Evolution 67:2753–61). Based on
 previous results (Dalla et al. 2017) the buffer regime used was 50 mM
 NaCl, 4 mM MgCl<sub>2</sub>, 50 mM imidazol, 20 mM KCl, pH
 7.4. The inhibition of the different Na,K-ATPases was tested under
 increasing ouabain and calotropin concentrations and expressed as
 percentage activity of a non-inhibited control. For each α/β-subunit
 construct technical duplicates of three biological replicates of
 recombinant proteins isolated from independently transfected
 <i>Sf</i>9 cells were tested.