Portal de Periódicos da CAPES

Structural Basis for α1 Versus α2 Isoform-distinct Behavior of the Na,K-ATPase

2003; Elsevier BV; Volume: 278; Issue: 11 Linguagem: Inglês

10.1074/jbc.m211636200

1083-351X

Laura Segall, Zahid Z. Javaid, Stephanie L. Carl, Lois K. Lane, Rhoda Blostein,

ATP Synthase and ATPases Research

We showed earlier that the kinetic behavior of the α2 isoform of the Na,K-ATPase differs from the ubiquitous α1 isoform primarily by a shift in the steady-state E 1 / E 2 equilibrium of α2 in favor of E 1 form(s). The aim of the present study was to identify regions of the α chain that confer the α1/α2 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 α2 that modulate conformational shifts are fundamentally similar to those of α1, suggesting that the predilection of α2 for E 1 state(s) is due to differences in primary structure of the two isoforms. Kinetic behavior of the α1/α2 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 α1/α2 divergent portion within the M4-M5 loop near the ATP binding domain. In addition, we show that the E 1 shift of α2 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).