Functional consequences of a posttransfection mutation in the H2-H3 cytoplasmic loop of the alpha subunit of Na,K-ATPase.

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 G950 to A substitution, which resulted in the replacement of glutamate at 233 with a lysine (E233K). E233K and α1 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 α1 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 μM ATP K+ inhibits Na+-ATPase of α1, but activates Na+-ATPase of E233K. This distinctive behavior of E233K is due to its faster rate of formation of dephosphoenzyme (E1) from K+-occluded enzyme (E2(K)), as well as 6-fold higher affinity for ATP at the low affinity ATP binding site. A lower ratio of Vmax to maximal level of phosphoenzyme indicates that E233K has a lower catalytic turnover than α1. These distinct kinetics of E233K suggest a shift in its E1/E2 conformational equilibrium toward E1. 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 Glu233 → Lys mutation.

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)(8)(9)(10)(11)(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 highly charged amino terminus had a role as a cation gate (13) now seems unlikely. Instead, interaction of this region with other region(s) of the ␣ protein affects the K ϩ deocclusion pathway of the reaction, probably via alteration in E 2 /E 1 conformational equilibrium. Our recent study (14) demonstrated that the highly charged sequence comprising residues 24 -32 of the amino terminus of the ␣ subunit is important in modulating the K ϩ deocclusion pathway of the reaction cycle. Furthermore, this modulation is not due to the 24 -32 nanopeptide per se, but rather to its interaction(s) with other isoform-specific region(s). This paper describes the characterization of a spontaneous, posttransfection mutation in the H2-H3 cytoplasmic loop of the ␣1 isoform of the Na,K-ATPase. It differs from the wild type enzyme by having a lysine substituted for glutamic acid in position 233, and is designated E233K. The mutant enzyme is functional and its apparent affinities for sodium and potassium are unaltered. However, it exhibits a decrease in catalytic turnover, an increase in apparent affinity for ATP, and an increase in the rate of conversion of E 2 (K) to E 1 .

EXPERIMENTAL PROCEDURES
Recovery and Analysis of Mutant E233K from HeLa Genomic DNA-Two sets of synthetic oligonucleotide primers were prepared for polymerase chain reaction amplification of the 5Ј and 3Ј halves of a putative spontaneous mutant in the NH 2 -terminal chimeric mutant of rat ␣1 cDNA (␣1(1-14␣2) cDNA) that had been incorporated into the HeLa genomic DNA in a pRc/CMV (Invitrogen) construct. The 5Ј primer set included a 32-mer complementary to the sequence of the sense strand of the T7 promoter of pRc/CMV and a 33-mer complementary to the antisense sequence between bases 1828 -1860 of the rat ␣1 cDNA. The 3Ј primer set included a 28-mer complementary to the sense strand of rat ␣1 between bases 1684 and 1711 and a 32-mer complementary to an antisense sequence in the SP6 promoter of pRc/CMV at the 3Ј end of the rat cDNA. The 5Ј and 3Ј halves of the ␣1(1-14␣2) cDNA were amplified from 1 g of HeLa genomic DNA utilizing these primer sets (10 pmol of each primer) and the TaqPlus polymerase reaction kit (Stratagene). Aliquots of the amplified DNA, with or without digestion with HindIII and BamHI, were analyzed on 0.8% agarose gels and compared to products obtained by amplification of 1 ng of purified pRc/CMV-rat ␣1 plasmid DNA. An aliquot of each reaction mixture was then utilized as template for additional amplification with the same primer sets. Amplified DNA from the 5Ј and 3Ј halves of rat ␣1(1-14␣2) was digested with HindIII and BamHI, gel-purified, and ligated into M13mp18/19 that had been digested with HindIII and BamHI. Competent DH5␣FЈ Escherichia coli were transformed with each ligation mixture and plated, and the resultant plaques were picked and amplified in liquid cultures. Aliquots of heat-treated supernatants of the M13 cultures were adjusted to 25 mM EDTA, 5% glycerol, and 0.4% SDS and electrophoresed on 0.7% agarose gels to identify M13 clones containing the ␣1 DNA inserts. Aliquots of the heat-treated supernatants of 37 ␣5Ј-containing clones and of 28 ␣3Ј-containing clones were pooled and used to infect DH5␣FЈ for the preparation of single-stranded M13mp18 -5Ј␣ and M13mp19 -3Ј␣ DNA. The 5Ј and 3Ј halves of the rat ␣1(1-14␣2) DNA from the pooled clones were completely sequenced using the Sanger dideoxy method (15), Sequenase version 2.0 (Amersham), and synthetic oligonucleotides as primers.
After detection of a single base substitution at base 950 in the pooled M13mp18 -5Ј␣ DNA, individual clones of M13mp18 -5Ј␣ were sequenced to identify a clone that contained the G 950 3 A nucleotide substitution and no other base changes between SalI(875) and BamHI(1780). Double-stranded DNA was then prepared from the identified M13mp18 -5Ј␣ clone, and a SalI(875)-BamHI(1870) fragment containing the G 950 3 A substitution was isolated and ligated into both the rat ␣1 wild type and the rat ␣1(1-14␣2) cDNAs in a modified pIBI30 shuttle vector, in place of the wild type SalI-BamHI fragments. The presence of the mutant sequences and the termini of the exchanged cassettes of these shuttle vector constructs were verified by DNA sequencing. The full-length ␣1 cDNAs were then isolated by HindIII digestion, gel-purified, and ligated into the HindIII site of pRc/CMV, and their orientations determined by restriction analyses. Twenty g of QIAGEN-purified expression plasmid DNA was used to transfect HeLa cells with CaPO 4 , as described by Chen and Okayama (16). HeLa cells expressing the mutant rat ␣1 proteins were selected by their ability to grow in medium containing 1 M ouabain, and clones were isolated and amplified as described previously (17).
Membrane Preparations and Enzyme Assays-Membranes were isolated, and assays of Na,K-ATPase, Na-ATPase, and ouabain-sensitive K ϩ (Rb ϩ ) influx were carried out as described previously (18). Assays of K ϩ -occluded enzyme and the formation of E 1 from K ϩ -occluded enzyme are also described elsewhere (14).

RESULTS
During the course of studies with a rat ␣1/␣2 NH 2 -terminal chimeric mutant cDNAs transfected into HeLa cells, we observed that the Na,K-ATPase of one clone had functional properties distinct from rat ␣1. The observation that the enzymic properties of several other clones selected from the same chimeric cDNA (rat ␣1(1-14␣2)) transfection were indistinguish-able from those of ␣1 suggested that a spontaneous, posttranfection mutation had occurred within the coding region of the rat ␣1 cDNA. As described under "Experimental Procedures," the alteration was identified as the substitution of lysine for glutamate at position 233 in the cytoplasmic region between transmembrane helices H2 and H3. The functional difference between this mutant (E233K) and the rat ␣1 enzyme was apparent when pump-mediated ATP hydrolysis was measured at micromolar ATP, under which condition ouabain-sensitive Na ϩ -dependent ATPase activity of the ␣1 isoform is inhibited by K ϩ (19). Accordingly, although K ϩ stimulates the dephosphorylation step of the reaction (E 2 P ϩ K 3 E 2 (K) ϩ P i ) and becomes occluded within the pump protein, its deocclusion is extremely slow. Stimulation of deocclusion is effected by ATP binding with low affinity (E 2 (K) ϩ ATP 3 ATP⅐E 1 ϩ K ϩ ; Ref. 19). As a consequence, K ϩ is an inhibitor of the overall reaction at low ATP, and an activator at high ATP concentration. In fact, as shown previously (14,18,20) and described below, at micromolar ATP the response of Na-ATPase to K ϩ is a sensitive means of characterizing isoform-or mutant-specific differences in the K ϩ deocclusion pathway of the reaction.
K ϩ Sensitivity of E233K-As shown in Fig. 1, the Na-ATPase activity of membranes isolated from rat ␣1-transfected cells is inhibited by the addition of 0.1-5 mM KCl, whereas the activity of E233K is markedly stimulated. At 1 mM KCl, the Na-ATPase activities of the mutant and ␣1 enzymes are 200% and 50%, respectively, of their control activities measured in the absence of KCl.
The change in K ϩ sensitivity at low ATP concentration caused by the mutation is suggestive of a change in rate of a reaction step following dephosphorylation of the K ϩ -sensitive form of phosphoenzyme commonly referred to as E 2 P in the Albers-Post model. According to a branched pathway of K ϩ deocclusion (Scheme I), it is assumed that ATP can bind with either (i) low affinity to the K ϩ -occluded form of the enzyme, E 2 (K), at a step preceding the release of potassium and the formation of ATP⅐E 1 (pathway a), or (ii) with high affinity at a step following the slow release of potassium from E 2 (K) (pathway b). Accordingly, potassium inhibition is dependent upon the apparent affinity of the enzyme for ATP at its low affinity site and/or the rate of release of K ϩ from E 2 (K). Analysis of kinetic parameters of pathways a and b is described below.
Kinetic Analysis of the Reaction Modeled According to ATP Binding to Low and High Affinity Sites-The kinetic parameters KЈ ATP , the apparent affinity for ATP, and V max for pathways a and b were obtained from measurements of Na,K-ATPase activity versus ATP concentration varied from 0.1 to 500 M. Reciprocal plots of the data are shown in Fig. 2, and the kinetic parameters are given in Table I. In the case of the ␣1 enzyme, the data points can be fitted to a two-component sys- tem, each linear in the ranges of 1-8 M and 25-500 M ATP. The constants for the apparent affinities for ATP at low and high affinity sites, designated KЈ L and KЈ H , respectively, are 331 Ϯ 44 M and 5.44 Ϯ 1.9 M for ␣1; V max of the high affinity component, V H , is 0.684 Ϯ 0.20 mol/(mg ϫ h) and represents 4.2% of the activity of the V max of the low affinity component, V L (16.2 Ϯ 3.0 mol/(mg ϫ h)). These values for ␣1 are similar to those reported previously (14). In contrast, the reciprocal plot of the E233K mutant is a straight line within the entire range of 1-500 M, resulting in an apparent affinity of 56.3 Ϯ 14 M and a V max (VЈ L ) of 9.90 Ϯ 1.1 mol/(mg ϫ h). Although these values are taken from representative experiments and there is some variability in V max values, it was observed that the activity of membranes prepared from E233K-transfected cells is generally lower than that of the rat ␣1-transfected cells.
An additional component of activity was observed in the range of 0.1-0.5 M ATP (data not shown), which represented Ϸ1-2% of V L . The constant for its apparent affinity for ATP, KЈ VH , was similar for both enzymes (0.19 Ϯ 0.09 M for ␣1 and 0.21 Ϯ 0.01 M for E233K). As suggested previously (14), this very high affinity component may represent an alternate minor pathway of ATP hydrolysis, involving K ϩ release directly from E 2 (K), i.e. E 2 (K) 3 E 2 ϩ K ϩ followed by the conversion of E 2 to E 1 . The nature of this minor component was not investigated further.
K ϩ Occlusion/Deocclusion-The marked difference in effect of K ϩ on Na-ATPase of E233K and ␣1 as depicted in Fig. 1 can be attributed to the higher affinity of E233K for ATP (lower KЈ L ) and/or higher rate of K ϩ deocclusion from E 2 (K) via pathway b. Since the low specific activity of the Na,K-ATPase expressed in HeLa cell membranes prevents the direct measurement of E 2 (K) and, therefore, the K ϩ deocclusion pathway of the reaction, an indirect method was used as described recently (14). In this assay, formation of the K ϩ -occluded enzyme is reflected by the decrease in phosphoenzyme (E 32 P) formed by phosphorylation with [␥-32 P]ATP following equilibration of the enzyme at room temperature (i) without and (ii) with K ϩ . The reduction in E 32 P resulting from preincubation with K ϩ (⌬E 32 P) is a measure of the amount of E 2 (K).
As shown in Fig. 3, and in agreement with our previous studies (14), the maximum formation of E 2 (K) with the ␣1 isoform is achieved after preincubation of the enzyme with 1 mM KCl, whereas with the ␣1E233K mutant maximum E 2 (K) requires at least 4 mM KCl. Using a simple model, , to describe the binding as in earlier studies of K ϩ occlusion in the kidney enzyme (21), the values of K 0.5 obtained are 1.0 Ϯ 0.5 and 0.12 Ϯ 0.03 mM for the E233K and ␣1 enzymes, respectively (mean Ϯ S.D. of three experiments). Maximum levels of E 2 (K), expressed as a percentage of phosphoenzyme formed without K ϩ preincubation (enzyme preincubated without KCl minus the KCl base line) were 93% Ϯ 10% and 97% Ϯ 5% for E233K and ␣1, respectively.
The shift in the equilibrium E 1 ϩ K ϩ 7 E 2 (K) toward E 1 can be accounted for by either a slower rate of occlusion or a faster rate of deocclusion. Although the rate of formation of E 1 from E 2 (K) is not a direct measure of K ϩ release from E 2 (K), it is characterized by single exponential kinetics (14) and, as a first approximation, is an estimate of the rate of deocclusion through pathway b.
In the representative experiments shown in Fig. 4, the rate of E 1 formation from E 2 (K) was determined as follows: ␣1 and E233K enzymes were first equilibrated with 8 mM KCl to form E 2 (K). ⌬E 32 P, the difference ((E 32 P formed following preincubation in the absence of K ϩ ) minus (E 32 P formed following preincubation with 8 mM K ϩ )) was taken to represent 100% K ϩ -occluded enzyme (Fig. 3). Deocclusion was measured by following the rate of increase in ⌬E 32 P at 10°C, a temperature at which deocclusion is sufficiently slow to permit manual assays. We showed previously that (i) maximal E 32 P formed from E 1 was similar at 10°C and 0°C, and remained constant over the period used to follow deocclusion at 10°C, and that ⌬E 32 P remained constant for up to 30 s at 0°C. Therefore, the time course of increase in ⌬E 32 P at 10°C reflects the rate of the rate-limiting step in the sequence E 2 (K) 3 E 1 K 3 E 1 at that temperature.
As can be seen in Fig. 4, the rate of formation of E 1 P from E 2 (K) is significantly faster for the E233K mutant compared to the ␣1 enzyme; rate constants are (s Ϫ1 ) 0.09 Ϯ 0.005 and 0.02 Ϯ 0.004 for E233K and ␣1, respectively. In other control experiments carried out with ␣1 and E233K, deocclusion was allowed to proceed for 10 s at 10°C in ATP-free Na ϩ medium, after which the E 1 formed was measured by rapid dilution and conversion to E 1 P at 0°C (5-s phosphorylation at 0°C). The rate constants estimated from the 10-s decrease in E 2 (K) were similar to those obtained with 1 M [␥-32 P]ATP present during deocclusion. This result indicates that the presence of 1 M ATP during deocclusion did not significantly affect the rate of deocclusion under these conditions. Phosphoenzyme Turnover and the Effect of Oligomycin-Since oligomycin stabilizes Na ϩ occlusion in E 1 (22,23) and   traps the enzyme in the E 1 P state, the extent to which EP increases in the presence of oligomycin is a measure of the steady-state distribution of the dephospho-and phosphoenzyme during steady-state catalysis. As shown in Table II (see  legend), phosphoenzyme measured at 0°C in the presence of oligomycin is increased 2.4-fold in the ␣1 enzyme and only 1.4-fold in E233K. Estimates of turnover, calculated as the ratio of V max to EP measured in the presence of oligomycin and Na ϩ , indicate that the E233K mutation results in a 40% reduction in turnover at 37°C. Sensitivity to pH-As described by Forbush and Klodos (24), the pH-dependence of Na,K-ATPase is limited partly by the rate of K ϩ deocclusion at acidic pH, by the rate of the E 1 P 3 E 2 P transition at neutral pH, and by phosphorylation at pH above pH 8.0. The pH dependence profiles of the E233K mutant and ␣1 enzymes shown in Fig. 5 indicate that as pH is lowered from the optimum at pH Ϸ 8.0 (room temperature) to pH 6.2, the decrease in activity was less for E233K (20% decrease) than for ␣1 (50% decrease). In contrast, the activity profile on the alkaline side of the optimum was similar for both enzyme forms.
Apparent Cation Affinities-To determine if the substitution of glutamate 233 with a lysine residue alters the apparent affinity of the mutant enzyme for cations, we compared the transport behavior and Na,K-ATPase kinetics of HeLa cells transfected with the E233K and ␣1 enzymes as described previously (18). Briefly, Rb 86 influx sensitive to high (10 mM) ouabain was measured in medium containing either various concentrations of sodium in the presence of monensin to control and maintain intracellular sodium concentration and saturating extracellular KCl concentration, or various concentrations of extracellular KCl, in the presence of monensin and 10 mM NaCl as described previously (25). We found that the apparent affinities for cytoplasmic Na ϩ (Na cyt ) of the E233K mutant and ␣1 enzymes are indistinguishable. In five separate experiments with the data fitted to a three-site cooperative model, the apparent affinities for Na cyt (K 0.5 ) are (mM) 20.9 Ϯ 2.0 for E233K and 20.1 Ϯ 2.7 for ␣1. Since intracellular Na ϩ could not be decreased below Ϸ15 mM with monensin present (25), we also compared the apparent affinities for Na ϩ in assays of Na,K-ATPase at 1 mM ATP and constant (10 mM) K ϩ . No Formation of E 32 P was measured at the indicated times following occlusion of K ϩ as described under "Experimental Procedures." Data are presented as percent of control E 32 P, which is the difference ((E 32 P formed in the absence of K ϩ 0°C) minus (E 32 P formed in the presence of 8 mM K ϩ at 10°C))/((E 32 P formed in the absence of K ϩ 0°C) minus (E 32 P formed in the presence of 8 mM K ϩ at 0°C)). Symbols are as described in the legend to  As shown earlier by Eisner and Richards (26) in studies of Na,K-ATPase of red cell ghosts, an increase in K ext results in an increase in KЈ ATP , and increasing the concentration of ATP leads to an increase in KЈ Kext . Although there is no difference in apparent affinities for either K ext or Na cyt between ␣1 and E233K pumps under the conditions of the flux assays carried out under physiological conditions of ATP concentration (K 0.5 values (mM) were 0.38 Ϯ 0.04 for E233K and 0.37 Ϯ 0.02 for ␣1 (average of five experiments), it is predicted that as the concentration of ATP is decreased below saturation, the apparent affinity for K ϩ for both enzymes would increase, and that the increase would be less for E233K than for ␣1. This prediction held true as shown by the analysis of K ϩ activation of Na,K-ATPase carried out at 1.0 and 0.1 mM ATP. At 1 mM ATP, with the data fitted to a two-site cooperative model, K 0.5 values (mM) for K ϩ are 1.11 Ϯ 0.034 and 1.76 Ϯ 0.12 for ␣1 and E233K, respectively; at 0.1 mM ATP, the K 0.5 values (mM) are 0.410 Ϯ 0.026 and 1.31 Ϯ 0.074 for ␣1 and E233K, respectively.
Vanadate Sensitivity-Vanadate interacts with the phosphorylation site in the catalytic H4-H5 domain and inhibits P-type ATPase. Studies with the yeast H ϩ -ATPase have provided evidence that structural alterations in the H2-H3 cytoplasmic loop result in changes in vanadate sensitivity (27,28). To assess interaction of Glu 233 with the catalytic phosphorylation site and/or the role of the region of this mutation on conformational coupling in the Na,K-ATPase, the effect of vanadate was tested. The results shown in Fig. 6 indicate that the Glu 233 3 Lys mutation causes a dramatic decrease in sensitivity to vanadate, with K i values for ␣1 and E233K being 8 and 2000 M, respectively.
Although the insensitivity to vanadate conferred by the Glu 233 3 Lys mutation is consistent with the conclusion that this change reflects an alteration in the steady-state E 1 /E 2 distribution as suggested for the yeast H ϩ -ATPase (27,28), other interpretations are plausible. The effect of this mutation is reminiscent of the decrease in vanadate sensitivity observed in the absence compared to presence of K ϩ (29), which raises the possibility of a direct alteration in enzyme-K ϩ interaction.
Another possibility is that binding of phosphate is altered. The former is less likely according to the argument that changes in KЈ K effected by mutations in cytoplasmic domains are probably secondary to changes in KЈ ATP as discussed below. DISCUSSION The experiments described in this report provide evidence for a functional role of the H2-H3 cytoplasmic loop in conformation coupling in the Na,K-ATPase. The changes effected by mutation of glutamate 233 to lysine are consistent with the conclusion that this substitution alters the equilibrium between the major conformational states of the dephospho-and phosphoforms during steady-state catalysis. An increase in the steadystate distribution of E 1 and E 2 in favor of E 1 effected by the Glu 233 3 Lys mutation is apparent as (i) an Ϸ4-fold increase in the rate constant for E 1 formation from E 2 (K), accounting for the higher K m for K ϩ occlusion of E233K compared to ␣1, (ii) a 6-fold increase in the apparent affinity for ATP at the step E 2 (K) ϩ ATP 3 ATP⅐E 1 ϩ K ϩ when the overall Na,K-ATPase reaction is studied at 37°C, and (iii) a lesser decrease in activity as pH is lowered to pH 6.8 under which condition deocclusion of K ϩ from E 2 (K) becomes the main rate-limiting reaction in the wild type enzyme (24). The results are also consistent with a shift in the E 1 P 7 E 2 P equilibrium toward E 1 P caused by the Glu 233 3 Lys mutation. The evidence is two-fold: the smaller increase in E 32 P effected by oligomycin and lower turnover of E233K compared to ␣1, at least at physiological pH under which condition the E 1 3 E 2 P transition is partly ratelimiting (24).
Glu 233 is in a region previously identified as having a role in the E 1 P 3 E 2 P conformational transition (for review, see Ref. 30). When Na,K-ATPase in the E 1 conformation is exposed to cleavage at either Leu 266 or Arg 262 in the H2-H3 cytoplasmic loop, as described by Jorgensen and co-workers (reviewed in Ref. 31), the E 1 P 3 E 2 P conformational transition is blocked. In the sarcoplasmic reticulum Ca-ATPase, tryptic cleavage at Arg 198 as well as site-specific mutation of residues in the predicted ␤-strand domain of the H2-H3 loop also blocked the E 1 P 3 E 2 P conformational transition (reviewed in Ref. 32).
As pointed out by Green and Stokes (33), the H2-H3 loop is well conserved among P-type ATPases. In their model of P-type ion pumps, based largely on studies of the sarcoplasmic reticulum Ca-ATPase (34), their suggestion that the anti-parallel ␤-strand is positioned close to the phosphorylation site is supported by vanadate protection of Ca-ATPase from proteolytic degradation (35). In the yeast H ϩ -ATPase, Perlin and co-workers (27,28) have shown that perturbations of residues in this region not only alters the distribution of conformational intermediates during steady-state catalysis, but also decreases the sensitivity of this P-type pump to vanadate inhibition, consistent also with the conclusion that this region interacts with the catalytic phosphorylation domain.
In studies of Na,K-ATPase, involvement of the H2-H3 loop in structural rearrangements associated with ligand binding and phosphorylation has been apparent in distinctive conformational changes revealed by proteolytic cleavage patterns (36). The importance of these interactions in conformation coupling is emphasized by the remarkable vanadate insensitivity caused by the Glu 233 3 Lys mutation. This vanadate insensitivity suggests interaction of Glu 233 located in the putative ␤-strand region of the H2-H3 loop with the phosphate binding domain within the catalytic H4-H5 loop. It is also possible that the vanadate insensitivity of E233K is the consequence of a decrease in steady-state level of E 2 required for P i (vanadate) binding (cf. the vanadate-insensitive mutants of PMA1; Ref. 28).
When deocclusion of K ϩ from the K ϩ -occluded state, E 2 (K), is analyzed as a branched pathway reaction (Scheme I; cf. Refs. 14 and 37), values of Na,K-ATPase activity as a function of varying ATP concentration can be fitted to a biphasic reciprocal plot in the case of the ␣1 enzyme. The ratio of V max values for the high affinity to low affinity components, V H /V L , is 0.043 for ␣1. In contrast, there is little, if any, evidence of a high affinity component (V H ) (pathway b) with the E233K enzyme. This is not unexpected, since, according to the simple Michaelis-Menten relationship shown in Equation 1, activity attributed to pathway a, compared to activity attributed to pathway b, is much greater in the E233K enzyme than it is in the ␣1 enzyme when ATP is reduced to micromolar concentrations.
With the E233K mutant, the 6-fold decrease in KЈ L results in a similar -fold increase in the rate of K ϩ deocclusion through pathway a, thus largely masking the activity through pathway b. Nevertheless, an increase in the rate of E 2 (K) 3 3 E 1 ϩ K ϩ is also observed with E233K under conditions in which E 2 (K) is first formed by equilibrating the enzyme with a saturating concentration of K ϩ . The increased rate of K ϩ deocclusion during subsequent incubation at 10°C also accounts for the higher K 0.5 for K ϩ under equilibrium binding conditions. It should be noted that the contribution of pathway b to overall activity may increase as temperature is increased. Under the conditions of the deocclusion assays, the relative rate of deocclusion through pathway a compared to that through b at 10°C is one-half that at 37°C. 1 It is of interest to compare this mutation with those in other cytoplasmic regions of the ␣1 subunit. In the H4-H5 loop, mutation of aspartate at the phosphorylation site of the Torpedo californica, pig, or sheep kidney enzymes results in complete loss of Na,K-ATPase activity (38 -40), and the latter two groups reported an increase in ATP affinity. Mutation of Lys 507 of the T. californica enzyme at the putative ATP binding site decreases both activity and ouabain binding capacity (38), and substitution of Cys 513 in the rat ␣1 enzyme decreases ATP affinity (41). In contrast to these mutations, a deletion mutant in which residues 1-32 have been removed from the cytoplasmic amino terminus of ␣1 results in a fully active enzyme with kinetic changes that are similar to those observed with the E233K mutation (14). This mutation results in a 2.5-fold increase in affinity for ATP at its low affinity binding site and an increase in rate through pathway b (14). With this mutant (␣1M32), as with E233K, the rate of conversion of E 2 (K) to E 1 and the apparent affinity for K ϩ occlusion were increased, and turnover was substantially reduced. Similarly, a NH 2 -terminal truncated mutant of the Bufo marinus enzyme was recently shown to have a reduced E 1 to E 2 conformational change (42). As shown elsewhere, the ␣2 isoform behaves similarly to ␣1M32 and may be regarded as a conformational variant of ␣1.
These cytoplasmic mutants contrast with those in which substitutions in transmembrane helices also result in active, functionally altered enzymes, most of which are characterized by changes in affinities for Na ϩ and K ϩ . Of these, one mutation localized to H5 (Glu 779 3 Gln; Ref. 11) alters (decreases) only Na ϩ affinity. An Asn 326 3 Leu mutation in H4 decreased the apparent affinity for Na ϩ but slightly increased it for K ϩ (8). Another group of transmembrane substitutions resulted in decreases in affinities for both Na ϩ and K ϩ . The substituted residues are, in H4: Glu 329 3 Gln (43) and Glu 327 3 Leu (9); in H5: Glu 781 3 Ala (7, 12), with quantitatively smaller changes noted in Glu 781 3 Asp (12); and in H6: Thr 809 3 Ala (7). In contrast to these mutations, substitution of Ser 775 in H5 with either alanine or cysteine decreases apparent K ϩ affinity dramatically, 31-fold in the case of Ser 775 3 Ala and 13-fold in the case of Ser 775 3 Cys, with no evidence of a change in Na ϩ affinity (10).
Substitutions in transmembrane helices effecting an increase in KЈ K are also associated with an increase in apparent affinity for ATP. In the analysis of Eisner and Richards (26) mentioned earlier, pump-mediated K ϩ influx was empirically described by a relationship which showed that decreasing K ext concentration (presumably equivalent to increasing KЈ Kext ), increases the apparent affinity for ATP. Similarly, increasing ATP concentration (presumably equivalent to decreasing KЈ ATP ), decreases the apparent affinity for external K ϩ (and also increases V max ). Considering, for example, the following sequence of reactions that constitute the low affinity pathway of K ϩ transport, it is likely that the various transmembrane residues which coordinate K ϩ ions probably do so with distinct affinities and/or selectivities.
Accordingly, distinct mutations may differentially alter the rate of specific reaction steps involved in K ϩ binding, occlusion, and deocclusion, with associated secondary changes in apparent affinity for ATP. The relationships between cation and ATP affinities of the cytoplasmic mutations are clearly different from those of mutations in transmembrane helices. Although the replacement of glutamate with lysine in the present study and, to a lesser extent, the deletion of the amino terminus described earlier (␣1M32) also increased the apparent affinity for ATP, changes in KЈ K were not observed as long as the ATP concentration was saturating. This behavior is consistent with the interpretation that the primary functional alteration effected by mutating Glu 233 to lysine is an increase in the rate of E 1 formation from E 2 (K). We suggest that this substitution in the cytoplasmic H2-H3 loop alters its interaction(s) with other regions of the ␣ subunit resulting in a change in the conformational equilibria, such that the apparent affinity for ATP is increased. Changes in apparent affinity for K ϩ that were observed at suboptimal ATP concentration are probably secondary to changes in KЈ ATP , as well as to steps involved in the conversion of conformational forms.