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.

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...

Previous studies showed that the ␣1, ␣2, and ␣3 isoforms of the catalytic subunit of the Na,K-ATPase differ in their apparent affinities for the ligands ATP, Na The experiments described in the present study provide insight into the mechanistic basis for these differences. The results show that ␣2 differs from ␣1 primarily by a shift in the E 1 º E 2 equilibrium in favor of E 1 form(s) as evidenced by (i) a ϳ20-fold increase in IC 50 for vanadate, (ii) decreased catalytic turnover, and (iii) notable stability of Na,K-ATPase activity at acidic pH. In contrast, despite its lower K ATP compared with ␣1, the E 1 º E 2 poise of ␣3 is not shifted toward E 1 . Distinct intrinsic interactions with Na ؉ ions are underscored by the marked selectivity for Na ؉ over Li ؉ of ␣3 compared with either ␣1 or ␣2 and higher K Na for cytoplasmic Na ؉ , which persists over a 100-fold range in proton concentration, independent of the presence of K ؉ . The kinetic analysis also suggests ␣3-specific differences in relative rates of partial reactions, which impact this isoform's distinct apparent affinities for both Na ؉ and K ؉ .

During kinetic studies of mutant rat Na,K-ATPases, we identified a spontaneous mutation in the first cytoplasmic loop between transmembrane helices 2 and 3 (H2-H3 loop) which results in a functional enzyme with distinct Na,K-ATPase kinetics. The mutant cDNA contained a single G 950 to A substitution, which resulted in the replacement of glutamate at 233 with a lysine (E233K). E233K and ␣1 cDNAs were transfected into HeLa cells and their kinetic behavior was compared. Transport studies carried out under physiological conditions with intact cells indicate that the E233K mutant and ␣1 have similar apparent affinities for cytoplasmic Na ؉ and extracellular K ؉ . In contrast, distinct kinetic properties are observed when ATPase activity is assayed under conditions (low ATP concentration) in which the K ؉ deocclusion pathway of the reaction is rate-limiting. At 1 M ATP K ؉ inhibits Na ؉ -ATPase of ␣1, but activates Na ؉ -ATPase of E233K. This distinctive behavior of E233K is due to its faster rate of formation of dephosphoenzyme (E 1 ) from K ؉ -occluded enzyme (E 2 (K)), as well as 6-fold higher affinity for ATP at the low affinity ATP binding site. A lower ratio of V max to maximal level of phosphoenzyme indicates that E233K has a lower catalytic turnover than ␣1. These distinct kinetics of E233K suggest a shift in its E 1 /E 2 conformational equilibrium toward E 1 . Furthermore, the importance of the H2-H3 loop in coupling conformational changes to ATP hydrolysis is underscored by a marked (2 orders of magnitude) reduction in vanadate sensitivity effected by this Glu 233 3 Lys mutation.The Na,K-ATPase is an integral membrane protein complex that catalyzes the exchange of three cytoplasmic sodium ions for two extracellular potassium ions coupled to the hydrolysis of one molecule of ATP. It is a heterodimeric protein comprising a catalytic ␣ subunit and a smaller heavily glycosylated ␤ subunit (for reviews, see Refs. 1 and 2). Although an additional protein, ␥, has been found associated with the ␣ and ␤ subunits (3), its function is unknown. The Na,K-ATPase is a member of the family of P-type ATPases, which are characteristically phosphorylated by ATP, and the phosphoenzyme intermediate undergoes rapid turnover during the reaction cycle. The phospho-as well as dephosphoenzymes exist in at least two distinct conformational states.During transport both sodium and potassium ions are occluded within the Na,K-ATPase (for review, see Ref. 4). The nature of the cation occlusion site(s) and the structures involved in gating of these sites to both the cytoplasmic and extracellular milieu are unknown. Approaches using chemical labeling (5, 6) and site-directed mutagenesis (7-12) have identified a number of functionally important carboxyl-containing amino acid residues in transmembrane regions H4, H5, H6, H8, and H9. Point mutations of putative cation binding amino acid residues that resulted in the expression of functional enzymes were characterized by an altered affinity for ATP, K ϩ , and/or Na ϩ . The prediction that the hi...

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