Mutation of Aspartate 804 of Na+,K+-ATPase Modifies the Cation Binding Pocket and Thereby Generates a High Na+-ATPase Activity

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Abstract
A series of six different mutants (D804A, D804E, D804G, D804N, D804Q, and D804S) of aspartate 804 present in transmembrane segment 6 of the rat Na+,K+-ATPase α1-subunit were prepared and expressed in Sf9 cells by use of the baculovirus expression system. In contrast to the wild-type enzyme all mutants except D804Q showed a very high Na+-ATPase activity, which was hardly further stimulated by the addition of K+. The ATPase activity of the mutants was already nearly maximal at 10 μM ATP and most of them could be phosphorylated in the absence of Na+ at pH 6.0 and 21 °C, suggesting that they strongly prefer the E1 over the E2 conformation. However, Na+ dose-dependently lowered the steady-state phosphorylation level, as a consequence of the increased affinity for Na+ in the dephosphorylation reaction of the mutants compared to the wild-type enzyme. Conversely, the affinity for K+ in the dephosphorylation reaction was decreased for the mutants as compared to that for the wild-type enzyme. When the pH was increased or the temperature was decreased, the phosphorylation level of the mutants decreased and the Na+ activation in the phosphorylation reaction became apparent. It is concluded that upon mutation of aspartate 804 the affinity of the cation-binding pocket is changed relatively in favor of Na+ instead of K+, as a consequence of which the enzyme has obtained a preference for the E1 conformation.