Mutations comprising either deletion of 32 amino acids from the NH 2 terminus (␣1M32) or a Glu 233 3 Lys substitution in the first M2-M3 cytoplasmic loop (E233K) of the ␣1-subunit of the Na,K-ATPase result in a shift in the steady-state E 1 7 E 2 conformational equilibrium toward E 1 form(s). In the present study, the functional consequences of both NH 2 -terminal deletion and Glu 233 substitution provide evidence for mutual interactions of these cytoplasmic regions. Following transfection and selection of HeLa cells expressing the ouabain-resistant ␣1M32E233K double mutant, growth was markedly reduced unless the K ؉ concentration in the culture medium was increased to at least 10 mM. Marked changes effected by this double mutation included 1) a 15-fold reduction in catalytic turnover (V max /EP max ), 2) a 70-fold increase in apparent affinity for ATP, 3) a marked decrease in vanadate sensitivity, and 4) marked (Ϸ10-fold) K ؉ activation of the Na-ATPase activity measured at micromolar ATP under which condition the E 2 (K) 3 3 E 1 pathway is normally (␣1) rate-limiting and K ؉ is inhibitory. The decrease in catalytic turnover was associated with a 5-fold decrease in V max and a compensatory Ϸ3-fold increase in expressed ␣1M32E233K protein. In contrast to the behavior of either ␣1M32 or E233K, ␣1M32E233K also showed alterations in apparent cation affinities. K Na was decreased Ϸ2-fold and K K was increased Ϸ2-fold. The importance of the charge at residue 233 is underscored by the consequences of single and double mutations comprising either a conservative change (E233D) or neutral substitution (E233Q). Thus, whereas mutation to a positively charged residue (E233K) causes a drastic change in enzymatic behavior, a conservative change causes only a minor change and the neutral substitution, an intermediate effect. Overall, the combined effects of the NH 2 -terminal deletion and the Glu 233 substitutions are synergistic rather than additive, consistent with an interaction between the NH 2 -terminal region, the first cytoplasmic loop, and possibly the large M4-M5 cytoplasmic loop bearing the nucleotide binding and phosphorylation sites.The Na,K-ATPase couples the hydrolysis of one ATP molecule to the translocation of 3 Na ϩ and 2 K ϩ ions against their electrochemical gradients, thus maintaining the normally high K ϩ and low Na ϩ concentrations inside animal cells. This enzyme complex comprises a large subunit, ␣ (molecular mass, 112 kDa) and a small subunit,  (molecular mass, 35 kDa). ␣ and  have been cloned and sequenced from a variety of tissues (see Ref. 1). The functional unit may be a heterodimer (␣) 2 , although a monomeric ␣ unit can occlude both Na ϩ and (K ϩ )Rb ϩ , consistent with its being the minimal unit required for transport (2). ␣ is the catalytic subunit, which spans the membrane probably 10 times and includes the cytoplasmic catalytic domain and the extracellular cardiac glycoside binding site(s) (3). Although this enzyme complex has eluded efforts to obtain ordered three-dimensional crystals o...
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...
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