We showed earlier that the kinetic behavior of the ␣2 isoform of the Na,K-ATPase differs from the ubiquitous ␣1 isoform primarily by a shift in the steady-state E 1 /E 2 equilibrium of ␣2 in favor of E 1 form(s). The aim of the present study was to identify regions of the ␣ chain that confer the ␣1/␣2 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 ؉ ) 3 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 3 E 2 P. We first confirmed that interactions between the cytoplasmic domains of ␣2 that modulate conformational shifts are fundamentally similar to those of ␣1, suggesting that the predilection of ␣2 for E 1 state(s) is due to differences in primary structure of the two isoforms. Kinetic behavior of the ␣1/␣2 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 ␣1/␣2 divergent portion within the M4-M5 loop near the ATP binding domain. In addition, we show that the E 1 shift of ␣2 results primarily from differences in the conformational transition of the dephosphoenzyme, (E 2 (K ؉ ) 3 E 1 ؉ K ؉ ), rather than phosphoenzyme (E 1 P 3 E 2 P).The Na,K-ATPase or sodium pump is an integral membrane protein complex found in the plasma membrane of virtually all animal cells. It catalyzes the exchange of three intracellular Na ϩ ions for two extracellular K ϩ ions using the energy of hydrolysis of one molecule of ATP. Consequently, the sodium pump plays an essential role in the maintenance of the electrochemical alkali cation gradients, providing the driving force for the transport of various nutrients into the cell. The Na,KATPase is a member of the family of P-type ATPases, which during the course of their catalytic cycle undergo phosphorylation and dephosphorylation of a conserved aspartate residue located in the large catalytic loop between transmembrane segments 4 and 5 of the catalytic ␣ subunit. During the catalytic cycle both dephospho-and phosphoenzymes undergo conformational transitions commonly referred to as E 1 7 E 2 and E 1 P 7 E 2 P, respectively. In addition to the large catalytic ␣ subunit, the Na,K-ATPase comprises a smaller, highly glycosylated ␤ subunit that is important for the proper folding of ␣ and its insertion into the plasma membrane. At present, four isoforms of ␣ and three isoforms of ␤ have been described, and these are distributed in a tissue-and developmentally dependent manner.The ␣2 isoform is located primarily in skeletal muscle and in brain, predominantly in glial cells. Our earlier studies indicated that it differs from t...