Structural basis for alpha1 versus alpha2 isoform-distinct behavior of the Na,K-ATPase.

We showed earlier that the kinetic behavior of the alpha2 isoform of the Na,K-ATPase differs from the ubiquitous alpha1 isoform primarily by a shift in the steady-state E(1)/E(2) equilibrium of alpha2 in favor of E(1) form(s). The aim of the present study was to identify regions of the alpha chain that confer the alpha1/alpha2 distinct behavior using a mutagenesis and chimera approach. Criteria to assess shifts in conformational equilibrium included (i) K(+) sensitivity of Na-ATPase measured at micromolar ATP, under which condition E(2)(K(+)) --> E(1) + K(+) becomes rate-limiting, (ii) changes in K'(ATP) for low affinity ATP binding, (iii) vanadate sensitivity of Na,K-ATPase activity, and (iv) the rate of the partial reaction E(1)P --> E(2)P.

New insights into the role of the N terminus in conformational transitions of the Na,K-ATPase.

The deletion of 32 residues from the N terminus of the alpha1 catalytic subunit of the rat Na,K-ATPase (mutant alpha1M32) shifts the E(1)/E(2) conformational equilibrium toward E(1), and the combination of this deletion with mutation E233K in the M2-M3 loop acts synergistically to shift the conformation further toward E(1) (Boxenbaum, N., Daly, S. E.