The alpha2 isoform of the Na,K-ATPase exhibits kinetic behavior distinct from that of the alpha1 isoform. The distinctive behavior is apparent when the reaction is carried out under conditions (micromolar ATP concentration) in which the K+ deocclusion pathway of the reaction cycle is rate-limiting; the alpha1 activity is inhibited by K+, whereas alpha2 is stimulated. When 32 NH2-terminal amino acid residues are removed from alpha1, the kinetic behavior of the mutant enzyme (alpha1M32) is similar to that of alpha2 (Daly, S. E., Lane, L. K., and Blostein, R. (1994) J. Biol. Chem. 269, 23944-23948). In the current study, the region of the alpha1 NH2 terminus involved in modulating this kinetic behavior has been localized to the highly charged sequence comprising residues 24-32. Within this nonapeptide, differences between alpha1 and alpha2 are conservative and are confined to residues 25-27. The behavior of two chimeric enzymes: (i) alpha1 with the first 32 residues identical to the alpha2 sequence, alpha1 (1-32alpha2), and (ii) alpha2 with the first 32 residues identical to the alpha1 sequence, alpha2(1-32alpha1), indicates that the distinctive kinetic behavior of alpha1 and alpha2 is not due to the 24-32 NH2-terminal domain, per se, but rather to its interaction with other, isoform-specific region(s) of the alpha1 protein. We also demonstrate that the distinct K+ activation profiles of either alpha2 or alpha1M32, compared to alpha1 is due to a faster release of K+ from the K+-occluded enzyme, and to a higher affinity for ATP. This was determined in studies using two approaches: (i) kinetic analysis of the reaction modeled according to a branched pathway of K+ deocclusion through low and high affinity ATP pathways and, (ii) measurements of the (rapid) phosphorylation of the enzyme (E1 conformation) by [gamma-32P]ATP following the rate-limiting formation of the K+-free enzyme from the K+-occluded state (E2(K) --> E1 + K+). The observed kinetic differences between alpha2 and alpha1 suggest that these Na,K-ATPase isoforms differ in the steady-state distribution of E1 and E2 conformational states.

The sodium/potassium-dependent ATPase [(Na+ + K+)ATPase], which establishes and maintains the Na+ and K+ gradients across the plasma membrane of animal cells, consists of two subunits, alpha and beta. Complementary DNA clones encoding the catalytic (alpha) subunit of sheep kidney and Torpedo californica electroplax enzymes have previously been isolated and characterized. However, there is little information concerning the primary structure of the beta-subunit, a glycoprotein of unknown function and relative molecular mass (Mr) approximately 55,000 (ref. 3). Here we describe the isolation and characterization of a cDNA clone containing the entire coding region of the beta-subunit of the sheep kidney (Na+ + K+)ATPase. We also discuss structural aspects of the protein and present evidence for a possible evolutionary relationship with the KdpC subunit of the Escherichia coli K+-ATPase.

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The deletion of 32 residues from the N terminus of the ␣1 catalytic subunit of the rat Na,K-ATPase (mutant ␣1M32) 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., Javaid, Z. Z., Lane, L. K., and Blostein, R. (1998) J. Biol. Chem. 273, 23086 -23092). To delimit the region of the cytoplasmic N terminus involved in these interactions, the consequences of a series of N-terminal deletions of ␣1 beyond ⌬32 were evaluated. Criteria to assess shifts in conformational equilibrium were based on effects of perturbation of the entire catalytic cycle ((i) sensitivity to vanadate inhibition, (ii) K ؉ sensitivity of Na-ATPase measured at micromolar ATP, (iii) changes in K ATP , and (iv) catalytic turnover), as well as estimates of the rates of the conformational transitions of phospho-and dephosphoenzyme (E 1 P 3 E 2 P and E 2 (K ؉ ) 3 E 1 ؉ K ؉ ). The results show that, compared with ␣1M32, the deletion of up to 40 residues (␣1M40) further shifts the poise toward E 1 . Remarkably, further deletions (mutants ␣1M46, ␣1M49, and ␣1M56) reverse the effect, such that these mutants increasingly resemble the wild type ␣1. These results suggest novel intramolecular interactions involving domains within the N terminus that impact the manner in which the N terminus/ M2-M3 loop regulatory domain interacts with the M4-M5 catalytic loop to effect E 1 7 E 2 transitions.The Na,K-ATPase or sodium pump is a ubiquitous integral membrane protein that catalyzes the glycoside sensitive, ATPcoupled exchange of three intracellular Na ϩ for two extracellular K ϩ ions across the plasma membrane of all animal cells. The sodium pump is essential to the maintenance of the electrochemical gradients of Na ϩ and K ϩ across the cell membrane, providing the driving force for the transport of nutrients into the cell and maintaining the cellular resting membrane potential. It comprises two essential subunits: a large catalytic ␣ subunit (ϳ100 kDa), containing the ligand binding and phosphorylation sites and a smaller, highly glycosylated ␤ subunit (ϳ35-55 kDa), which acts as a chaperone for ␣ (for review see Refs. 1 and 2). A third subunit, ␥ (ϳ7 kDa), was found in the kidney where it functions as a regulator of the pump (see Refs. 3 and 4).The sodium pump is a member of a family of transporters known as P-type ATPases that are directly phosphorylated and dephosphorylated on a conserved aspartate residue within their cytoplasmic domain during the course of the reaction cycle. Both the phosphorylated and dephosphorylated forms of the enzyme can exist in at least two states that undergo conformational transitions (E 1 P 3 E 2 P and E 2 3 E 1 ) that are coupled to the ion-translocating steps. Definitive evidence for distinct E 1 and E 2 conformational states and a role of the N terminus in effecting E 1 7 E 2 transitions was first obtained in 1975 by Jorgensen (5, 6) using tryptic cleav...

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...