The C Terminus of Na+,K+-ATPase Controls Na+ Affinity on Both Sides of the Membrane through Arg935
2009; Elsevier BV; Volume: 284; Issue: 28 Linguagem: Inglês
10.1074/jbc.m109.015099
ISSN1083-351X
AutoresMads S. Toustrup-Jensen, Rikke Holm, Anja P. Einholm, Vivien R. Schack, Jens Preben Morth, Poul Nissen, Jens Peter Andersen, Bente Vilsen,
Tópico(s)Plant Stress Responses and Tolerance
ResumoThe Na+,K+-ATPase C terminus has a unique location between transmembrane segments, appearing to participate in a network of interactions. We have examined the functional consequences of amino acid substitutions in this region and deletions of the C terminus of varying lengths. Assays revealing separately the mutational effects on internally and externally facing Na+ sites, as well as E1-E2 conformational changes, have been applied. The results pinpoint the two terminal tyrosines, Tyr1017 and Tyr1018, as well as putative interaction partners, Arg935 in the loop between transmembrane segments M8 and M9 and Lys768 in transmembrane segment M5, as crucial to Na+ activation of phosphorylation of E1, a partial reaction reflecting Na+ interaction on the cytoplasmic side of the membrane. Tyr1017, Tyr1018, and Arg935 are furthermore indispensable to Na+ interaction on the extracellular side of the membrane, as revealed by inability of high Na+ concentrations to drive the transition from E1P to E2P backwards toward E1P and inhibit Na+-ATPase activity in mutants. Lys768 is not important for Na+ binding from the external side of the membrane but is involved in stabilization of the E2 form. These data demonstrate that the C terminus controls Na+ affinity on both sides of the membrane and suggest that Arg935 constitutes an important link between the C terminus and the third Na+ site, involving an arginine-π stacking interaction between Arg935 and the C-terminal tyrosines. Lys768 may interact preferentially with the C terminus in E1 and E1P forms and with the loop between transmembrane segments M6 and M7 in E2 and E2P forms. The Na+,K+-ATPase C terminus has a unique location between transmembrane segments, appearing to participate in a network of interactions. We have examined the functional consequences of amino acid substitutions in this region and deletions of the C terminus of varying lengths. Assays revealing separately the mutational effects on internally and externally facing Na+ sites, as well as E1-E2 conformational changes, have been applied. The results pinpoint the two terminal tyrosines, Tyr1017 and Tyr1018, as well as putative interaction partners, Arg935 in the loop between transmembrane segments M8 and M9 and Lys768 in transmembrane segment M5, as crucial to Na+ activation of phosphorylation of E1, a partial reaction reflecting Na+ interaction on the cytoplasmic side of the membrane. Tyr1017, Tyr1018, and Arg935 are furthermore indispensable to Na+ interaction on the extracellular side of the membrane, as revealed by inability of high Na+ concentrations to drive the transition from E1P to E2P backwards toward E1P and inhibit Na+-ATPase activity in mutants. Lys768 is not important for Na+ binding from the external side of the membrane but is involved in stabilization of the E2 form. These data demonstrate that the C terminus controls Na+ affinity on both sides of the membrane and suggest that Arg935 constitutes an important link between the C terminus and the third Na+ site, involving an arginine-π stacking interaction between Arg935 and the C-terminal tyrosines. Lys768 may interact preferentially with the C terminus in E1 and E1P forms and with the loop between transmembrane segments M6 and M7 in E2 and E2P forms. The Na+,K+-ATPase is a membrane-bound ion pump that uses energy liberated by hydrolysis of ATP to exchange intracellular Na+ for extracellular K+ at a ratio of 3:2, thus creating the gradients for Na+ and K+ across the cell membrane required for electrical excitability, cellular uptake of ions, nutrients, and neurotransmitters, and regulation of cell volume and intracellular pH (1.Glynn I.M. J. Physiol. 1993; 462: 1-30Crossref PubMed Scopus (119) Google Scholar, 2.Kaplan J.H. Annu. Rev. Biochem. 2002; 71: 511-535Crossref PubMed Scopus (922) Google Scholar). Like other P-type ATPases, such as the Ca2+-ATPase and H+,K+-ATPase, the Na+,K+-ATPase forms a phosphorylated enzyme intermediate through transfer of the γ-phosphate of ATP to a conserved aspartate residue in the P-domain. It is generally believed that the transport mechanism is consecutive, the Na+ ions being translocated before K+. The binding of three Na+ ions from the cytoplasmic side to the E1 form triggers phosphorylation of the enzyme by ATP, and binding of two K+ ions from the extracellular side to E2P activates the dephosphorylation ("Post-Albers model," Scheme 1) (3.Post R.L. Hegyvary C. Kume S. J. Biol. Chem. 1972; 247: 6530-6540Abstract Full Text PDF PubMed Google Scholar). During transfer across the membrane, the Na+ and K+ ions become occluded in the binding pocket, being temporarily unable to dissociate to either side of the membrane. The three Na+ ions are released sequentially to the external side in connection with the E1P → E2P conformational transition, and K+ dissociates to the cytoplasmic side in association with the E2 → E1 transition of the dephosphoenzyme (Scheme 1). Recently, the structure of the Na+,K+-ATPase in the E2 form with two occluded Rb+ ions bound as K+ congeners was determined by x-ray crystallography at 3.5 Å resolution (4.Morth J.P. Pedersen B.P. Toustrup-Jensen M.S. Sørensen T.L. Petersen J. Andersen J.P. Vilsen B. Nissen P. Nature. 2007; 450: 1043-1049Crossref PubMed Scopus (717) Google Scholar). The α-subunit consists of 10 membrane-spanning helices, αM1–αM10, 2The abbreviations used are: αM1–αM10 or M1–M10the 10 transmembrane segments of the α-subunit numbered from the N terminusβMthe transmembrane segment of the β-subunit. and three distinct cytoplasmic domains denoted A (actuator), N (nucleotide binding), and P (phosphorylation) by analogy to the closely related sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) (5.Toyoshima C. Nakasako M. Nomura H. Ogawa H. Nature. 2000; 405: 647-655Crossref PubMed Scopus (1636) Google Scholar). The K+ binding pocket is located between transmembrane helices αM4, αM5, αM6, and αM8. The K+ binding sites are comprised of residues homologous to those binding the two Ca2+ ions in the E1 form of the Ca2+-ATPase, and it is therefore likely that they also make up two of the three Na+ binding sites in the Na+,K+-ATPase E1 form. The location of the third Na+ site that has no counterpart in the Ca2+-ATPase remains elusive (4.Morth J.P. Pedersen B.P. Toustrup-Jensen M.S. Sørensen T.L. Petersen J. Andersen J.P. Vilsen B. Nissen P. Nature. 2007; 450: 1043-1049Crossref PubMed Scopus (717) Google Scholar). The Na+,K+-ATPase also consists of, in addition to the α-subunit, a β-subunit with a large extracellular domain and a single membrane span (βM). The β-subunit is required for routing the α-subunit to the plasma membrane and is furthermore essential for K+ binding (6.Lutsenko S. Kaplan J.H. Biochemistry. 1993; 32: 6737-6743Crossref PubMed Scopus (127) Google Scholar), possibly because it influences the positioning of some of the transmembrane segments of the α-subunit in E2 conformation. In many cell types, the αβ-complex interacts with a small regulatory transmembrane protein belonging to the FXYD family (7.Geering K. Am. J. Physiol. Renal. Physiol. 2006; 290: F241-F250Crossref PubMed Scopus (295) Google Scholar). Because the Na+ concentration in a normal cell is rather low (typically around 10–15 mm), the Na+,K+-ATPase operates at only a fraction of its maximal pumping rate. Hence, changes in the cytoplasmic Na+ concentration or in the affinity for Na+ constitute important regulatory measures, and the Na+ affinity is differentially affected by the various FXYD proteins. the 10 transmembrane segments of the α-subunit numbered from the N terminus the transmembrane segment of the β-subunit. The crystal structure of the Na+,K+-ATPase (4.Morth J.P. Pedersen B.P. Toustrup-Jensen M.S. Sørensen T.L. Petersen J. Andersen J.P. Vilsen B. Nissen P. Nature. 2007; 450: 1043-1049Crossref PubMed Scopus (717) Google Scholar) revealed an unexpected location of the C terminus of the α-subunit between the transmembrane helices (see Fig. 1A). The C terminus consists of a PGG motif followed by an extension of eight residues relative to the corresponding C terminus of the Ca2+-ATPase (SERCA1a isoform). The first part of this extension forms a small helix between the transmembrane helices βM, αM7, and αM10, and the two C-terminal tyrosine residues are accommodated in a binding pocket between αM7, αM8, and αM5 (see Fig. 1, B–D). This unique position prompted us to examine the functional role of the C terminus by deletion of the five most C-terminal residues KETYY, which reduced the Na+ affinity of the E1 form conspicuously, thus indicating a previously unrecognized importance of the C terminus for Na+ binding at one or more of the three cytoplasmically facing activating sites (4.Morth J.P. Pedersen B.P. Toustrup-Jensen M.S. Sørensen T.L. Petersen J. Andersen J.P. Vilsen B. Nissen P. Nature. 2007; 450: 1043-1049Crossref PubMed Scopus (717) Google Scholar). The putative role of the C terminus in regulation of Na+ affinity led us to denote it "switch region." It was, however, not clear, whether the position of the C-terminal carboxylate group, the interaction of the side chains, or both are crucial for Na+ binding. The C terminus and its binding pocket are well resolved in the crystal structure, as indicated by the electron density map contoured at 1σ in Fig. 1, A and B, thus providing information of considerable detail and confidence. Hence, a possible involvement of the C-terminal carboxylate group as well as the hydroxyl groups of the two C-terminal tyrosines in hydrogen/salt bridge formation is suggested (see Fig. 1, B–D). The terminal tyrosine residue, 3All numbering of Na+,K+-ATPase residues in this article refers to the sequence of the rat α1-isoform. Tyr1018, seems to be in position to make bonds with Lys768 in M5 and Arg935 in the loop connecting M8 and M9, and Tyr1017 may also interact with Arg935 (Fig. 1, B–D). It is unknown whether these bonds suggested by the crystal structure of the E2 form with bound Rb+ exist in the Na+-bound E1 form and, if so, whether they could mediate the observed effects of the C terminus on Na+ affinity. An important question is furthermore whether the C terminus is of any importance in relation to the Na+ sites in the externally facing configuration in the phosphoenzyme. To understand in detail the importance of the structural features of the C terminus, we have in this study examined the functional consequences of replacing individual amino acids in this region and introducing deletions of the C terminus of variable length. The interaction with Na+ in the dephosphoenzyme as well as in the phosphoenzyme was studied, thus revealing separately the mutational effects on internally and externally exposed Na+ sites. Mutations were introduced into full-length cDNA encoding the rat kidney Na+,K+-ATPase (α1-isoform) using the QuikChange site-directed mutagenesis kit (Stratagene), and the mutants and wild type were expressed in COS-1 cells, using 5 μm ouabain in the growth medium to inhibit the endogenous COS-1 cell enzyme and thereby select stable transfectants (8.Vilsen B. FEBS Lett. 1992; 314: 301-307Crossref PubMed Scopus (52) Google Scholar, 9.Jewell E.A. Lingrel J.B. J. Biol. Chem. 1991; 266: 16925-16930Abstract Full Text PDF PubMed Google Scholar). The presence of the desired mutations was verified by sequencing the cDNA before transfection into the COS-1 cells, and in most cases, also by sequencing the DNA stably integrated into the genome of the isolated ouabain-resistant COS-1 cell lines. The crude plasma membrane fraction containing expressed wild-type or mutant Na+,K+-ATPase was isolated by differential centrifugation, and prior to functional analysis, the plasma membranes were made leaky with alamethicin or deoxycholate (8.Vilsen B. FEBS Lett. 1992; 314: 301-307Crossref PubMed Scopus (52) Google Scholar). Hence, it can be assumed that the membrane potential was zero during the measurements. Measurements of the ATPase activity by following the release of Pi at 37 °C and phosphorylation/dephosphorylation studies using [γ-32P]ATP were carried out as described previously (8.Vilsen B. FEBS Lett. 1992; 314: 301-307Crossref PubMed Scopus (52) Google Scholar, 10.Vilsen B. Biochemistry. 1997; 36: 13312-13324Crossref PubMed Scopus (49) Google Scholar, 11.Toustrup-Jensen M. Hauge M. Vilsen B. Biochemistry. 2001; 40: 5521-5532Crossref PubMed Scopus (28) Google Scholar, 12.Rodacker V. Toustrup-Jensen M. Vilsen B. J. Biol. Chem. 2006; 281: 18539-18548Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar); the media compositions are detailed in the figure legends. The acid-precipitated 32P-labeled phosphoenzyme was washed by centrifugation and subjected to SDS-polyacrylamide gel electrophoresis at pH 6.0 (10.Vilsen B. Biochemistry. 1997; 36: 13312-13324Crossref PubMed Scopus (49) Google Scholar), and the radioactivity associated with the separated Na+,K+-ATPase band was quantified by "imaging" using a Packard CycloneTM storage phosphor system. The active site concentration was determined by phosphorylation at 0 °C in the presence of 2 μm [γ-32P]ATP, 3 mm Mg2+, 150 mm Na+, and oligomycin (20 μg/ml) to block dephosphorylation and thereby obtain stoichiometric phosphorylation (10.Vilsen B. Biochemistry. 1997; 36: 13312-13324Crossref PubMed Scopus (49) Google Scholar). The catalytic turnover rate was calculated as the ratio between the rate of ATP hydrolysis and the active site concentration. The contribution from endogenous Na+,K+-ATPase in the functional assays was eliminated by including ouabain in the reaction media at a concentration sufficient to inhibit the endogenous enzyme but too low to affect the rat Na+,K+-ATPase. Each data point shown is the average value corresponding to at least three independent measurements. Data normalization, averaging, and non-linear regression analysis were carried out as described previously (10.Vilsen B. Biochemistry. 1997; 36: 13312-13324Crossref PubMed Scopus (49) Google Scholar, 11.Toustrup-Jensen M. Hauge M. Vilsen B. Biochemistry. 2001; 40: 5521-5532Crossref PubMed Scopus (28) Google Scholar), and the results are reported ± S.E. Seventeen new mutations to residues in the C-terminal region of the α-subunit were studied (Table 1). These mutations encompass C-terminal deletions of varying lengths ranging from one or both of the two terminal tyrosines to an eight-residue deletion including the complete C-terminal helix (WVEKE). The EKE part of the helix was also examined in a triple mutation replacing these three charged residues by alanines. The residues in the preceding loop, Pro1008, Gly1009, and Gly1010, were likewise examined by alanine substitutions. The two C-terminal tyrosines were examined by single and double replacements with alanine removing the aromatic function, and to study the importance of the phenolic hydroxyl groups, each of the tyrosines was replaced with phenylalanine. The functional importance of the putative interactions of Arg935 with the C terminus (Fig. 1) was examined by substitution of Arg935 with alanine, and the juxtaposed Arg936 was likewise studied by replacement with alanine. The M5 residue Lys768 within interaction distance of the C terminus was replaced with both alanine and methionine, the latter to remove the positive charge but retain the bulk of the side chain.TABLE 1Ligand concentration dependence and relative amount of E2PK0.5(K+)aData are from Fig. 2 or were obtained from similar experiments with the remaining mutants.K0.5(Na+)bData are from Fig. 3 or were obtained from similar experiments with the remaining mutants.E2PcThese values represent the amplitude of the slow phase corresponding to the fitted curves in Fig. 5; the Na+ concentration is 150 mm.K0.5(ATP)dData are from Fig. 8 or were obtained from similar experiments with the remaining mutants.K0.5(vanadate)eData are from Fig. 9 or were obtained from similar experiments with the remaining mutants.K0.5(ouabain)fThese values represent the K0.5 value for inhibition of the Na+,K+-ATPase activity by ouabain. The Na+,K+-ATPase activity was determined at 37 °C in 30 mm histidine buffer (pH 7.4), 130 mm NaCl, 20 mm KCl, 3 mm ATP, 3 mm MgCl2, 1 mm EGTA, and various concentrations of ouabain. A function with the ouabain-inhibited enzyme represented by the sum of two hyperbolic components (V = Vtot − a1[ouabain]/(K1 + [ouabain]) − a2[ouabain]/(K2 + [ouabain])), a high affinity component corresponding to endogenous COS-1 cell Na+,K+-ATPase and a low affinity component corresponding to recombinant exogenous rat Na+,K+-ATPase, was fitted to the data. The K0.5 value determined for the endogenous enzyme was ≤1.0 μm.μmμm%μmμmμmWild type669 ± 14437 ± 957360 ± 173.2 ± 0.1158 ± 7ΔY (delgdel, deletion. Tyr1018)878 ± 301016 ± 34NDhND, not determined.176 ± 94.3 ± 0.1124 ± 8ΔYY (del Tyr1017–Tyr1018)1021 ± 134059 ± 7875172 ± 83.6 ± 0.1140 ± 11ΔTYY (del Thr1016–Tyr1018)793 ± 347559 ± 23068104 ± 85.9 ± 0.5195 ± 37ΔKETYY (del Lys1014–Tyr1018)iΔKETYY has been previously studied (4), and the K0.5 values for Na+, ATP, and vanadate are the same as reported in Ref. (4).700 ± 4311,458 ± 63776114 ± 95.4 ± 0.3240 ± 46ΔWVEKETYY (del Trp1011–Tyr1018)695 ± 6810,854 ± 63385100 ± 86.0 ± 0.5255 ± 35Y1018A933 ± 172211 ± 49ND252 ± 121.8 ± 0.1118 ± 7Y1018F905 ± 13617 ± 18ND357 ± 373.3 ± 0.2144 ± 12Y1017A720 ± 281169 ± 55ND272 ± 172.0 ± 0.1151 ± 14Y1017F691 ± 12525 ± 16ND388 ± 292.3 ± 0.1147 ± 11YY-AA (Y1017A/Y1018A)555 ± 113,919 ± 61470145 ± 66.1 ± 0.2276 ± 10T1016A814 ± 27582 ± 25ND294 ± 115.0 ± 0.4144 ± 24EKE-AAA (E1013A/K1014A/E1015A)879 ± 31650 ± 2154220 ± 67.2 ± 0.20121 ± 4GG-AA (G1009A/G1010A)850 ± 45532 ± 2958446 ± 393.3 ± 0.2136 ± 17P1008A722 ± 35411 ± 15ND441 ± 282.9 ± 0.1155 ± 7R935A558 ± 52045 ± 7784299 ± 182.4 ± 0.2232 ± 17R936A660 ± 14647 ± 27ND321 ± 112.7 ± 0.1149 ± 8K768A430 ± 112105 ± 504540 ± 239.2 ± 1.4876 ± 52K768M722 ± 471449 ± 52ND120 ± 38.1 ± 0.3191 ± 10a Data are from Fig. 2 or were obtained from similar experiments with the remaining mutants.b Data are from Fig. 3 or were obtained from similar experiments with the remaining mutants.c These values represent the amplitude of the slow phase corresponding to the fitted curves in Fig. 5; the Na+ concentration is 150 mm.d Data are from Fig. 8 or were obtained from similar experiments with the remaining mutants.e Data are from Fig. 9 or were obtained from similar experiments with the remaining mutants.f These values represent the K0.5 value for inhibition of the Na+,K+-ATPase activity by ouabain. The Na+,K+-ATPase activity was determined at 37 °C in 30 mm histidine buffer (pH 7.4), 130 mm NaCl, 20 mm KCl, 3 mm ATP, 3 mm MgCl2, 1 mm EGTA, and various concentrations of ouabain. A function with the ouabain-inhibited enzyme represented by the sum of two hyperbolic components (V = Vtot − a1[ouabain]/(K1 + [ouabain]) − a2[ouabain]/(K2 + [ouabain])), a high affinity component corresponding to endogenous COS-1 cell Na+,K+-ATPase and a low affinity component corresponding to recombinant exogenous rat Na+,K+-ATPase, was fitted to the data. The K0.5 value determined for the endogenous enzyme was ≤1.0 μm.g del, deletion.h ND, not determined.i ΔKETYY has been previously studied (4.Morth J.P. Pedersen B.P. Toustrup-Jensen M.S. Sørensen T.L. Petersen J. Andersen J.P. Vilsen B. Nissen P. Nature. 2007; 450: 1043-1049Crossref PubMed Scopus (717) Google Scholar), and the K0.5 values for Na+, ATP, and vanadate are the same as reported in Ref. (4.Morth J.P. Pedersen B.P. Toustrup-Jensen M.S. Sørensen T.L. Petersen J. Andersen J.P. Vilsen B. Nissen P. Nature. 2007; 450: 1043-1049Crossref PubMed Scopus (717) Google Scholar). Open table in a new tab The mutations were introduced into the cDNA encoding the ouabain-resistant rat α1-isoform of the Na+,K+-ATPase, and the wild-type and mutant rat enzymes were expressed using the mammalian COS-1 cell system under ouabain-selective pressure (8.Vilsen B. FEBS Lett. 1992; 314: 301-307Crossref PubMed Scopus (52) Google Scholar, 9.Jewell E.A. Lingrel J.B. J. Biol. Chem. 1991; 266: 16925-16930Abstract Full Text PDF PubMed Google Scholar). All the mutants were capable of sustaining cell growth in the presence of ouabain at a concentration inhibiting the endogenous COS-1 cell Na+,K+-ATPase, indicating that the Na+ and K+ transport rates of the mutants are sufficiently high to be compatible with cell viability. The catalytic turnover rate (Na+,K+-ATPase activity per active site) measured at 130 mm Na+, 20 mm K+, and 3 mm ATP on the isolated membranes containing the expressed exogenous enzyme was markedly reduced for mutant K768A (2896 ± 179 min−1 (n = 8), compare with wild type 8474 ± 165 min−1 (n = 11)). A slight reduction relative to wild type was found for mutants R935A (6278 ± 311 min−1 (n = 8)) and ΔWVEKETYY (6556 ± 125 min−1 (n = 6)). For the remaining mutants there was no significant reduction of the catalytic turnover rate measured under these conditions (e.g. ΔYY 8059 ± 339 min−1 (n = 8) and YY-AA 9193 ± 614 min−1 (n = 8)). (See Table 1 for definitions of the multiple substitutions used in this study.) Fig. 2 shows the K+ concentration dependence of the Na+,K+-ATPase activity at a relatively low Na+ concentration of 40 mm. K+ present at submillimolar concentrations activates ATP hydrolysis due to the stimulation of dephosphorylation by K+ binding at the external sites on E2P (cf. Scheme 1). All the mutants showed wild type-like (less than 2-fold deviation from wild type) apparent affinity for K+ activation (Fig. 2 and Table 1). However, several of the mutants also displayed, in addition to the K+ activation phase, an inhibition phase at high K+ concentrations. This inhibition was particularly manifest (>20% inhibition) for the C-terminal deletion mutants ΔYY, ΔTYY, and ΔWVEKETYY as well as for YY-AA, R935A, and K768A. A less pronounced inhibition was seen for Y1017A, Y1018A, and K768M, whereas there was no inhibition of the wild type, ΔY, Y1017F, Y1018F, T1016A, R936A, P1008A, GG-AA, and EKE-AAA (Fig. 2). As demonstrated by Skou (13.Skou J.C. Biochim. Biophys. Acta. 1957; 23: 394-401Crossref PubMed Scopus (1316) Google Scholar) in his pioneering first study on the Na+,K+ pump, such an inhibition phase can also be detected for the wild-type enzyme if the Na+ concentration is lowered. This inhibition is caused by K+ binding in competition with Na+ at the cytoplasmically facing E1 sites. The inhibition seen here for some of the mutants may therefore be caused by an increased ability of K+ to compete with Na+ at the E1 sites. To determine the Na+ affinity at the cytoplasmically facing sites of the E1 form, the Na+ dependence of phosphorylation from [γ-32P]ATP was studied in the absence of K+, and in the presence of oligomycin added to stabilize the Na+-occluded form as much as possible (Fig. 3). A 32-fold reduction of the Na+ affinity relative to wild type was seen for YY-AA, and dramatic reductions were also found for the C-terminal deletion mutants ΔYY, ΔTYY, and ΔWVEKETYY (9-, 17-, and 25-fold, respectively, Fig. 3 and Table 1). The previously studied ΔKETYY mutant (4.Morth J.P. Pedersen B.P. Toustrup-Jensen M.S. Sørensen T.L. Petersen J. Andersen J.P. Vilsen B. Nissen P. Nature. 2007; 450: 1043-1049Crossref PubMed Scopus (717) Google Scholar), giving a 26-fold reduction of Na+ affinity for phosphorylation, fits nicely into this picture (data not shown here, but see summary in Table 1). It is noteworthy that the double substitution of the two terminal tyrosines with alanines, YY-AA, reduced the Na+ affinity significantly more than deletion of the two tyrosines. A 5-fold reduction of Na+ affinity was seen upon single alanine substitution of the most C-terminal tyrosine, Y1018A, whereas 2.7- and 2.3-fold reductions were seen for alanine substitution of the preceding tyrosine, Y1017A, and deletion of a single tyrosine, ΔY, respectively. Removal of the side chain hydroxyl group from each of the tyrosines by replacement with phenylalanine did not affect Na+ affinity significantly (mutants Y1017F and Y1018F), and the same was the case for the mutations T1016A, R936A, P1008A, GG-AA, and EKE-AAA, whereas R935A reduced Na+ affinity 5-fold. A 5-fold reduction was also seen for K768A, and a smaller, 3.3-fold reduction was seen for K768M (Fig. 3 and Table 1). These results correlate very well with the inhibitory effects of high K+ concentrations seen in Fig. 2, the mutants with the most pronounced inhibition by K+ also showing the largest reduction of Na+ affinity, thus indicating that the enhanced ability of K+ to compete with Na+ in the E1 form is caused by a weaker Na+ binding at one or more of the E1 sites. Because oligomycin stabilizes the Na+-occluded state (14.Esmann M. Skou J.C. Biochem. Biophys. Res. Commun. 1985; 127: 857-863Crossref PubMed Scopus (63) Google Scholar, 15.Skou J.C. Kaplan J.H. De Weer P. The Sodium Pump: Recent Developments. The Rockefeller University Press, New York1991: 317-319Google Scholar), thereby promoting phosphorylation and reducing dephosphorylation by blocking the E1P → E2P transition, the phosphoenzyme builds up to a maximal level in the presence of oligomycin. Fig. 4 shows the amount of phosphoenzyme accumulated in the absence of oligomycin, relative to the maximal phosphorylation level observed in the presence of oligomycin as stabilizer (EP/EPmax ratio), at two Na+ concentrations of 150 and 600 mm. For the wild type, the EP/EPmax ratio was 85 and 69% at 150 and 600 mm Na+, respectively, and for most mutants, EP/EPmax was wild type-like at 150 mm Na+. Only K768A showed a marked reduction of EP/EPmax to 37% under these conditions. At 600 mm Na+, the mutants ΔYY, ΔTYY, ΔWVEKETYY, YY-AA, R935A, K768A, and K768M all showed a conspicuous reduction of EP/EPmax to 20% or less. For comparison, we included the previously described ΔKETYY mutant (4.Morth J.P. Pedersen B.P. Toustrup-Jensen M.S. Sørensen T.L. Petersen J. Andersen J.P. Vilsen B. Nissen P. Nature. 2007; 450: 1043-1049Crossref PubMed Scopus (717) Google Scholar), and this mutant showed a reduction of the EP/EPmax ratio to the same low level at 600 mm Na+ (Fig. 4). The low EP/EPmax ratios may be the result of either a reduced phosphorylation rate or an increased dephosphorylation rate (see further under "Discussion"). The two phosphoenzyme intermediates, E1P and E2P, are distinguished by difference in reactivity with ADP. The E1P intermediate, which has three Na+ ions bound in an occluded state, is ADP-sensitive, i.e. able to react with ADP and donate the phosphoryl group back to ADP, forming ATP. By contrast, E2P is ADP-insensitive but dephosphorylates by hydrolysis of the aspartyl phosphoryl bond. The latter reaction is activated by K+, binding with high affinity at two extracellularly facing sites on E2P (Scheme 1), and to some extent by Na+, presumably binding at the same sites as K+ but with low affinity (3.Post R.L. Hegyvary C. Kume S. J. Biol. Chem. 1972; 247: 6530-6540Abstract Full Text PDF PubMed Google Scholar). Upon the addition of ADP to the phosphoenzyme formed from [γ-32P]ATP, two decay phases can be distinguished, a rapid phase corresponding to E1P reacting backward with ADP and a slow phase corresponding to decay of E2P (10.Vilsen B. Biochemistry. 1997; 36: 13312-13324Crossref PubMed Scopus (49) Google Scholar, 11.Toustrup-Jensen M. Hauge M. Vilsen B. Biochemistry. 2001; 40: 5521-5532Crossref PubMed Scopus (28) Google Scholar, 12.Rodacker V. Toustrup-Jensen M. Vilsen B. J. Biol. Chem. 2006; 281: 18539-18548Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Fig. 5 shows the results of such ADP dephosphorylation experiments carried out with selected mutants following phosphorylation with [γ-32P]ATP in the absence of oligomycin (to allow subsequent dephosphorylation) and in the presence of 150 mm Na+ (to obtain a sufficiently high phosphorylation level for the dephosphorylation rate to be accurately determined, cf. Fig. 4). The relative amounts of the rapidly and slowly dephosphorylating components were determined by fitting a biexponential function, and the amplitude of the slow phase (E2P) is indicated in Table 1. The mutants ΔYY, ΔTYY, ΔKETYY, ΔWVEKETYY, YY-AA, and R935A, displaying a large reduction in affinity for cytoplasmic Na+ (Fig. 3), also exhibited a higher E2P level (68–85%) than wild type (57%). Mutant K768A exhibited a lower E2P level (45%) than the wild type, i.e. an increased amount of ADP-sensitive E1P, and mutants EKE-AAA and GG-AA were wild type-like. Experiments measuring Na+ flux and ADP-ATP exchange using sided Na+,K+-ATPase membrane preparations have established that Na+ binds to intracellular activating sites as well as to extracellular inhibitory and activating sites (16.Garrahan P.J. Glynn I.M. J. Physiol. 1967; 192: 159-174Crossref PubMed Scopus (172) Google Scholar, 17.Glynn I.M. Karlish S.J.D. J. 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To examine whether the increased E2P fraction of the phosphoenzyme seen for s
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