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. We first confirmed that interactions between the cytoplasmic domains of alpha2 that modulate conformational shifts are fundamentally similar to those of alpha1, suggesting that the predilection of alpha2 for E(1) state(s) is due to differences in primary structure of the two isoforms. Kinetic behavior of the alpha1/alpha2 chimeras indicates that the difference in E(1)/E(2) poise of the two isoforms cannot be accounted for by their notably distinct N termini, but rather by the front segment extending from the cytoplasmic N terminus to the C-terminal end of the extracellular loop between transmembranes 3 and 4, with a lesser contribution of the alpha1/alpha2 divergent portion within the M4-M5 loop near the ATP binding domain. In addition, we show that the E(1) shift of alpha2 results primarily from differences in the conformational transition of the dephosphoenzyme, (E(2)(K(+)) --> E(1) + K(+)), rather than phosphoenzyme (E(1)P --> E(2)P).