Selective Fe2+-catalyzed oxidative cleavage of gastric H+,K+-ATPase: implications for the energy transduction mechanism of P-type cation pumps

In the presence of ascorbate/H(2)O(2), Fe(2+) ions or the ATP-Fe(2+) complex catalyze selective cleavage of the alpha subunit of gastric H(+),K(+)-ATPase. The electrophoretic mobilities of the fragments and dependence of the cleavage patterns on E(1) and E(2) conformational states are essentially identical to those described previously for renal Na(+),K(+)-ATPase. The cleavage pattern of H(+),K(+)-ATPase by Fe(2+) ions is consistent with the existence of two Fe(2+) sites: site 1 within highly conserved sequences in the P and A domains, and site 2 at the cytoplasmic entrance to trans-membrane segments M3 and M1. The change in the pattern of cleavage catalyzed by Fe(2+) or the ATP-Fe(2+) complex induced by different ligands provides evidence for large conformational movements of the N, P, and A cytoplasmic domains of the enzyme. The results are consistent with the Ca(2+)-ATPase crystal structure (Protein Data Bank identification code; Toyoshima, C., Nakasako, M., Nomura, H., and Ogawa, H. (2000) Nature 405, 647-655), an E(1)Ca(2+) conformation, and a theoretical model of Ca(2+)-ATPase in an E(2) conformation (Protein Data Bank identification code ). Thus, it can be presumed that the movements of N, P, and A cytoplasmic domains, associated with the E(1) <--> E(2) transitions, are similar in all P-type ATPases. Fe(2+)-catalyzed cleavage patterns also reveal sequences involved in phosphate, Mg(2+), and ATP binding, which have not yet been shown in crystal structures, as well as changes which occur in E(1) <--> E(2) transitions, and subconformations induced by H(+),K(+)-ATPase-specific ligands such as SCH28080.     

D443 of the N domain of Na+,K+-ATPase interacts with the ATP-Mg2+ complex, possibly via a second Mg2+ ion

This paper provides evidence for an interaction of D443 in the N domain of Na(+),K(+)-ATPase with a Mg(2+) ion. Wild-type, D443N/A/C and S445A mutants of porcine Na(+),K(+)-ATPase (alpha1beta1) have been expressed in Pichia pastoris. By comparison with wild-type, D443N reduces the turn-over rate by about 40%. Binding affinity of ATP, measured directly, was not affected by D443N, D443A, or D443C mutations. AMP-PNP-Fe(2+)-catalyzed oxidative cleavage of Na(+),K(+)-ATPase produces two characteristic fragments, at (708)VNDS (P domain) and near (440)VAGDA (N domain), respectively. In the D443N and D443A mutants, both cleavages are suppressed, indicating an interaction between the residues with AMP-PNP-Fe(2+) bound. Previous work suggested that with ATP-Fe(2+) bound the N and P domains come into proximity, both D710 and D443 making contact with a single Fe(2+) (or Mg(2+)) ion. However, the crystal structure of Ca(2+)-ATPase with bound AMP-PCP and Mg(2+) confirm the involvement of D703 (D710) but show that E439 (D443) is too far to make contact with the Mg(2+). By contrast, in the crystal structure with bound ADP, AlF(4), and Mg(2+), representing the E(1)-P conformation, two Mg(2+) ions were observed. Significantly, ADP-Fe(2+)-mediated oxidative cleavage of renal Na,K-ATPase produces the fragment near (440)VAGDA (N domain), while the cleavage at (708)VNDS (P domain) is almost completely absent. The results are explained economically by the hypothesis that ATP is bound with two Mg(2+) (Fe(2+)) ions, a "catalytic" Mg(2+) interacting with D710 via the gamma phosphate and a "structural" Mg(2+) interacting with D443 via the alpha and beta phosphates and a water molecule, respectively.     

The ATP-Mg2+ binding site and cytoplasmic domain interactions of Na+,K+-ATPase investigated with Fe2+-catalyzed oxidative cleavage and molecular modeling

This work utilizes Fe(2+)-catalyzed cleavages and molecular modeling to obtain insight into conformations of cytoplasmic domains and ATP-Mg(2+) binding sites of Na(+),K(+)-ATPase. In E(1) conformations the ATP-Fe(2+) complex mediates specific cleavages at 712VNDS (P domain) and near 440VAGDA (N domain). In E(2)(K), ATP-Fe(2+) mediates cleavages near 212TGES (A domain), near 440VAGDA, and between residues 460-490 (N domain). Cleavages at high ATP-Fe(2+) concentrations do not support suggestions for two ATP sites. A new reagent, fluorescein-DTPA, has been synthesized. The fluorescein-DTPA-Fe(2+) complex mediates cleavages similar to those mediated by ATP-Fe(2+). The data suggest the existence of N to P domain interactions in E(1)Na, with bound ATP-Fe(2+) or fluorescein-DPTA-Fe(2+), A-N, and A-P interactions in E(2)(K), and provide testable constraints for model building. Molecular models based on the Ca(2+)-ATPase structure are consistent with the predictions. Specifically, high-affinity ATP-Mg(2+) binding in E(1) is explained with the N domain tilted ca. 80 degrees toward the P domain, by comparison with well-separated N and P domains in the Ca-ATPase crystal structure. With ATP-Mg(2+) docked, bound Mg(2+) is close to both D710 (in 710DGVNDS) and D443 (in 440VAGDASE). D710 is known to be crucial for Mg(2+) binding. The cleavage and modeling data imply that D443 could also be a candidate for Mg(2+) binding. Comparison of E(1).ATP,Mg(2+) and E(2) models suggests an explanation of the high or low ATP affinities, respectively. We propose a scheme of ATP-Mg(2+) and Mg(2+) binding and N, P, and A domain interactions in the different conformations of the catalytic cycle.     

Dimethyl sulfoxide-induced conformational state of Na(+)/K(+)-ATPase studied by proteolytic cleavage

Effects of dimethyl sulfoxide (Me(2)SO) on substrate affinity for phosphorylation by inorganic phosphate, on phosphorylation by ATP in the absence of Na(+), and on ouabain binding to the free form of the Na(+)/K(+)-ATPase have been attributed to changes in solvation of the active site or Me(2)SO-induced changes in the structure of the enzyme. Here we used selective trypsin cleavage as a procedure to determine the conformations that the Na(+)/K(+)-ATPase acquires in Me(2)SO medium. In water or in Me(2)SO medium, Na(+)/K(+)-ATPase exhibited after partial proteolysis two distinct groups of fragments: (1) in the presence of 0.1 M Na(+) or 0.1 M Na(+) + 3 mM ADP (enzyme in the E1 state) cleavage produced a main fragment of about 76 kDa; and (2) in the presence of 20 mM K(+) (E2 state) a 58-kDa fragment plus two or three fragments of 39-41 kDa were obtained. Cleavage in Me(2)SO medium in the absence of Na(+) and K(+) exhibited the same breakdown pattern as that obtained in the presence of K(+), but a 43-kDa fragment was also observed. An increase in the K(+) concentration to 0.5 mM eliminated the 43-kDa fragment, while a 39- to 41-kDa doublet was accumulated. Both in water and in Me(2)SO medium, a strong enhancement of the 43-kDa band was observed in the presence of either P(i) + ouabain or vanadate, suggesting that the 43-kDa fragment is closely related to the conformation of the phosphorylated enzyme. These results indicate that Me(2)SO acts not only by promoting the release of water from the ATP site, but also by inducing a conformation closely related to the phosphorylated state, even when the enzyme is not phosphorylated.     

Entrance port for Na(+) and K(+) ions on Na(+),K(+)-ATPase in the cytoplasmic loop between trans-membrane segments M6 and M7 of the alpha subunit. Proximity Of the cytoplasmic segment of the beta subunit

Based on the following observations we propose that the cytoplasmic loop between trans-membrane segments M6 and M7 (L6/7) of the alpha subunit of Na(+),K(+)-ATPase acts as an entrance port for Na(+) and K(+) ions. 1) In defined conditions chymotrypsin specifically cleaves L6/7 in the M5/M6 fragment of 19-kDa membranes, produced by extensive proteolysis of Na(+),K(+)-ATPase, and in parallel inactivates Rb(+) occlusion. 2) Dissociation of the M5/M6 fragment from 19-kDa membranes is prevented either by occluded cations or by competitive antagonists such as Ca(2+), Mg(2+), La(3+), p-xylylene bisguanidinium and m-xylylene bisguanidinium, or 1-bromo-2,4, 6-tris(methylisothiouronium)benzene and 1,3-dibromo-2,4,6-tris (methylisothiouronium)benzene (Br(2)-TITU(3+)). 3) Ca(2+) ions raise electrophoretic mobility of the M5/M6 fragment but not that of the other fragments of the alpha subunit. It appears that negatively charged residues in L6/7 recognize either Na(+) or K(+) ions or the competitive cation antagonists. Na(+) and K(+) ions are then occluded within trans-membrane segments and can be transported, whereas the cation antagonists are not occluded and block transport at the entrance port. The cytoplasmic segment of the beta subunit appears to be close to or contributes to the entrance port, as inferred from the following observations. 1) Specific chymotryptic cleavage of the 16-kDa fragment of the beta subunit to 15-kDa at 20 degrees C (Shainskaya, A., and Karlish, S. J. D. (1996) J. Biol. Chem. 271, 10309-10316) markedly reduces affinity for Br(2)-TITU(3+) and for Na(+) ions, detected by Na(+) occlusion assays or electrogenic Na(+) binding, whereas Rb(+) occlusion is unchanged. 2) Na(+) ions specifically protect the 16-kDa fragment against this chymotryptic cleavage.     

Expression of Na+,K+-ATPase in Pichia pastoris: analysis of wild type and D369N mutant proteins by Fe2+-catalyzed oxidative cleavage and molecular modeling

Na+,K+-ATPase (pig alpha1,beta1) has been expressed in the methylotrophic yeast Pichia pastoris. A protease-deficient strain was used, recombinant clones were screened for multicopy genomic integrants, and protein expression, and time and temperature of methanol induction were optimized. A 3-liter culture provides 300-500 mg of membrane protein with ouabain binding capacity of 30-50 pmol mg-1. Turnover numbers of recombinant and renal Na+,K+-ATPase are similar, as are specific chymotryptic cleavages. Wild type (WT) and a D369N mutant have been analyzed by Fe2+- and ATP-Fe2+-catalyzed oxidative cleavage, described for renal Na+,K+-ATPase. Cleavage of the D369N mutant provides strong evidence for two Fe2+ sites: site 1 composed of residues in P and A cytoplasmic domains, and site 2 near trans-membrane segments M3/M1. The D369N mutation suppresses cleavages at site 1, which appears to be a normal Mg2+ site in E2 conformations. The results suggest a possible role of the charge of Asp369 on the E1 <--> E2 conformational equilibrium. 5'-Adenylyl-beta,gamma-imidodi-phosphate(AMP-PNP)-Fe2+-catalyzed cleavage of the D369N mutant produces fragments in P (712VNDS) and N (near 440VAGDA) domains, described for WT, but only at high AMP-PNP-Fe2+ concentrations, and a new fragment in the P domain (near 367CSDKTGT) resulting from cleavage. Thus, the mutation distorts the active site. A molecular dynamic simulation of ATP-Mg2+ binding to WT and D351N structures of Ca2+-ATPase (analogous to Asp369 of Na+,K+-ATPase) supplies possible explanations for the new cleavage and for a high ATP affinity, which was observed previously for the mutant. The Asn351 structure with bound ATP-Mg2+ may resemble the transition state of the WT poised for phosphorylation.     

Chimeras of X+, K+-ATPases. The M1-M6 region of Na+, K+-ATPase is required for Na+-activated ATPase activity, whereas the M7-M10 region of H+, K+-ATPase is involved in K+ de-occlusion

In this study we reveal regions of Na(+),K(+)-ATPase and H(+),K(+)-ATPase that are involved in cation selectivity. A chimeric enzyme in which transmembrane hairpin M5-M6 of H(+),K(+)-ATPase was replaced by that of Na(+),K(+)-ATPase was phosphorylated in the absence of Na(+) and showed no K(+)-dependent reactions. Next, the part originating from Na(+),K(+)-ATPase was gradually increased in the N-terminal direction. We demonstrate that chimera HN16, containing the transmembrane segments one to six and intermediate loops of Na(+),K(+)-ATPase, harbors the amino acids responsible for Na(+) specificity. Compared with Na(+),K(+)-ATPase, this chimera displayed a similar apparent Na(+) affinity, a lower apparent K(+) affinity, a higher apparent ATP affinity, and a lower apparent vanadate affinity in the ATPase reaction. This indicates that the E(2)K form of this chimera is less stable than that of Na(+),K(+)-ATPase, suggesting that it, like H(+),K(+)-ATPase, de-occludes K(+) ions very rapidly. Comparison of the structures of these chimeras with those of the parent enzymes suggests that the C-terminal 187 amino acids and the beta-subunit are involved in K(+) occlusion. Accordingly, chimera HN16 is not only a chimeric enzyme in structure, but also in function. On one hand it possesses the Na(+)-stimulated ATPase reaction of Na(+),K(+)-ATPase, while on the other hand it has the K(+) occlusion properties of H(+),K(+)-ATPase.     

Fe2+ -catalyzed oxidative cleavages of Ca2+ -ATPase reveal novel features of its pumping mechanism

We have analyzed the Fe2+ -catalyzed oxidative cleavages of Ca2+ -ATPase in the presence of Ca2+, with or without the ATP analog 5'-adenylyl-beta,gamma-imidodiphosphate (AMP-PNP) or in the presence of the inhibitor thapsigargin. To identify the positions of cleavages as precisely as possible, we have used previously identified proteinase K and tryptic fragments as a standard, advanced mass spectrometry techniques, as well as specific antibodies. A number of cleavages are similar to those described for Na+,K+ -ATPase or other P-type pumps and are expected on the basis of the putative Mg2+ binding residues near the phosphorylated Asp351 in E1 or E2P conformations. However, intriguing new features have also been observed. These include a Fe2+ site near M3, which cannot be due to the presence of histidine residues as it was postulated in the case of Na+,K+ -ATPase and H+,K+ -ATPase. This site could represent a Ca2+ binding zone between M1 and M3, preceding Ca2+ occlusion within M4, 5, 6, and 8. In addition, we present evidence that, in the non-crystalline state, the N- and P-domain may approach each other, at least temporarily, in the presence of Ca2+ (E1Ca2 conformation), whereas the presence of Mg.ATP stabilizes the N to P interaction (E1.Mg.ATP conformation).     

Mutations Phe785Leu and Thr618Met in Na+,K+-ATPase, associated with familial rapid-onset dystonia parkinsonism, interfere with Na+ interaction by distinct mechanisms

The Na(+),K(+)-ATPase plays key roles in brain function. Recently, missense mutations in the Na(+),K(+)-ATPase were found associated with familial rapid-onset dystonia parkinsonism (FRDP). Here, we have characterized the functional consequences of FRDP mutations Phe785Leu and Thr618Met. Both mutations lead to functionally altered, but active, Na(+),K(+)-pumps, that display reduced apparent affinity for cytoplasmic Na(+), but the underlying mechanism differs between the mutants. In Phe785Leu, the interaction of the E(1) form with Na(+) is defective, and the E(1)-E(2) equilibrium is not displaced. In Thr618Met, the Na(+) affinity is reduced because of displacement of the conformational equilibrium in favor of the K(+)-occluded E(2)(K(2)) form. In both mutants, K(+) interaction at the external activating sites of the E(2)P phosphoenzyme is normal. The change of cellular Na(+) homeostasis is likely a major factor contributing to the development of FRDP in patients carrying the Phe785Leu or Thr618Met mutation. Phe785Leu moreover interferes with Na(+) interaction on the extracellular side and reduces the affinity for ouabain significantly. Analysis of two additional Phe(785) mutants, Phe785Leu/Leu786Phe and Phe785Tyr, demonstrated that the aromatic function of the side chain, as well as its exact position, is critical for Na(+) and ouabain binding. The effects of substituting Phe(785) could be explained by structural modeling, demonstrating that Phe(785) participates in a hydrophobic network between three transmembrane segments. Thr(618) is located in the cytoplasmic part of the molecule near the catalytic site, and the structural modeling indicates that the Thr618Met mutation interferes with the bonding pattern in the catalytic site in the E(1) form, thereby destabilizing E(1) relative to E(2)(K(2)).     

Inhibition of Na+/K(+)-ATPase and Mg(2+)-ATPase by metal ions and prevention and recovery of inhibited activities by chelators

Kinetics and inhibition of Na(+)/K(+)-ATPase and Mg(2+)-ATPase activity from rat synaptic plasma membrane (SPM), by separate and simultaneous exposure to transition (Cu(2+), Zn(2+), Fe(2+) and Co(2+)) and heavy metals (Hg(2+) and Pb(2+)) ions were studied. All investigated metals produced a larger maximum inhibition of Na(+)/K(+)-ATPase than Mg(2+)-ATPase activity. The free concentrations of the key species (inhibitor, MgATP(2-), MeATP(2-)) in the medium assay were calculated and discussed. Simultaneous exposure to the combinations Cu(2+)/Fe(2+) or Hg(2+)/Pb(2+) caused additive inhibition, while Cu(2+)/Zn(2+) or Fe(2+)/Zn(2+) inhibited Na(+)/K(+)-ATPase activity synergistically (i.e., greater than the sum metal-induced inhibition assayed separately). Simultaneous exposure to Cu(2+)/Fe(2+) or Cu(2+)/Zn(2+) inhibited Mg(2+)-ATPase activity synergistically, while Hg(2+)/Pb(2+) or Fe(2+)/Zn(2+) induced antagonistic inhibition of this enzyme. Kinetic analysis showed that all investigated metals inhibited Na(+)/K(+)-ATPase activity by reducing the maximum velocities (V(max)) rather than the apparent affinity (Km) for substrate MgATP(2-), implying the noncompetitive nature of the inhibition. The incomplete inhibition of Mg(2+)-ATPase activity by Zn(2+), Fe(2+) and Co(2+) as well as kinetic analysis indicated two distinct Mg(2+)-ATPase subtypes activated in the presence of low and high MgATP(2-) concentration. EDTA, L-cysteine and gluthathione (GSH) prevented metal ion-induced inhibition of Na(+)/K(+)-ATPase with various potencies. Furthermore, these ligands also reversed Na(+)/K(+)-ATPase activity inhibited by transition metals in a concentration-dependent manner, but a recovery effect by any ligand on Hg(2+)-induced inhibition was not obtained.     

Identification of potential regulatory sites of the Na+,K+-ATPase by kinetic analysis

Kinetic models are presented that allow the Na(+),K(+)-ATPase steady-state turnover number to be estimated at given intra- and extracellular concentrations of Na(+), K(+), and ATP. Based on experimental transient kinetic data, the models utilize either three or four steps of the Albers-Post scheme, that is, E(2) --> E(1), E(1) --> E(2)P (or E(1) --> E(1)P and E(1)P --> E(2)P), and E(2)P --> E(2), which are the major rate-determining steps of the enzyme cycle. On the time scale of these reactions, the faster binding steps of Na(+), K(+), and ATP to the enzyme are considered to be in equilibrium. Each model was tested by comparing calculations of the steady-state turnover from rate constants and equilibrium constants for the individual partial reactions with published experimental data of the steady-state activity at varying Na(+) and K(+) concentrations. To provide reasonable agreement between the calculations and the experimental data, it was found that Na(+)/K(+) competition for cytoplasmic binding sites was an essential feature required in the model. The activity was also very dependent on the degree of K(+)-induced stimulation of the reverse reaction E(1) --> E(2). Taking into account the physiological substrate concentrations, the models allow the most likely potential sites of short-term Na(+),K(+)-ATPase regulation to be identified. These were found to be (a) the cytoplasmic Na(+) and K(+) binding sites, via changes in Na(+) or K(+) concentration or their dissociation constants, (b) ATP phosphorylation (as a substrate), via a change in its rate constant, and (c) the position of the E(2)<==>E(1) equilibrium.     

Chimeric domain analysis of the compatibility between H(+), K(+)-ATPase and Na(+),K(+)-ATPase beta-subunits for the functional expression of gastric H(+),K(+)-ATPase.

Gastric H(+),K(+)-ATPase consists of alpha-subunit with 10 transmembrane domains and beta-subunit with a single transmembrane domain. We constructed cDNAs encoding chimeric beta-subunits between the gastric H(+),K(+)-ATPase and Na(+),K(+)-ATPase beta-subunits and co-transfected them with the H(+),K(+)-ATPase alpha-subunit cDNA in HEK-293 cells. A chimeric beta-subunit that consists of the cytoplasmic plus transmembrane domains of Na(+),K(+)-ATPase beta-subunit and the ectodomain of H(+),K(+)-ATPase beta-subunit assembled with the H(+),K(+)-ATPase alpha-subunit and expressed the K(+)-ATPase activity. Therefore, the whole cytoplasmic and transmembrane domains of H(+),K(+)-ATPase beta-subunit were replaced by those of Na(+),K(+)-ATPase beta-subunit without losing the enzyme activity. However, most parts of the ectodomain of H(+),K(+)-ATPase beta-subunit were not replaced by the corresponding domains of Na(+), K(+)-ATPase beta-subunit. Interestingly, the extracellular segment between Cys(152) and Cys(178), which contains the second disulfide bond, was exchangeable between H(+),K(+)-ATPase and Na(+), K(+)-ATPase, preserving the K(+)-ATPase activity intact. Furthermore, the K(+)-ATPase activity was preserved when the N-terminal first 4 amino acids ((67)DPYT(70)) in the ectodomain of H(+),K(+)-ATPase beta-subunit were replaced by the corresponding amino acids ((63)SDFE(66)) of Na(+),K(+)-ATPase beta-subunit. The ATPase activity was abolished, however, when 4 amino acids ((76)QLKS(79)) in the ectodomain of H(+),K(+)-ATPase beta-subunit were replaced by the counterpart ((72)RVAP(75)) of Na(+),K(+)-ATPase beta-subunit, indicating that this region is the most N-terminal one that discriminates the H(+),K(+)-ATPase beta-subunit from that of Na(+), K(+)-ATPase.     

Identification and function of a cytoplasmic K+ site of the Na+, K+ -ATPase

A cytoplasmic nontransport K(+)/Rb(+) site in the P-domain of the Na(+), K(+)-ATPase has been identified by anomalous difference Fourier map analysis of crystals of the [Rb(2)].E(2).MgF(4)(2-) form of the enzyme. The functional roles of this third K(+)/Rb(+) binding site were studied by site-directed mutagenesis, replacing the side chain of Asp(742) donating oxygen ligand(s) to the site with alanine, glutamate, and lysine. Unlike the wild-type Na(+), K(+)-ATPase, the mutants display a biphasic K(+) concentration dependence of E(2)P dephosphorylation, indicating that the cytoplasmic K(+) site is involved in activation of dephosphorylation. The affinity of the site is lowered significantly (30-200-fold) by the mutations, the lysine mutation being most disruptive. Moreover, the mutations accelerate the E(2) to E(1) conformational transition, again with the lysine substitution resulting in the largest effect. Hence, occupation of the cytoplasmic K(+)/Rb(+) site not only enhances E(2)P dephosphorylation but also stabilizes the E(2) dephosphoenzyme. These characteristics of the previously unrecognized nontransport site make it possible to account for the hitherto poorly understood trans-effects of cytoplasmic K(+) by the consecutive transport model, without implicating a simultaneous exposure of the transport sites toward the cytoplasmic and extracellular sides of the membrane. The cytoplasmic K(+)/Rb(+) site appears to be conserved among Na(+), K(+)-ATPases and P-type ATPases in general, and its mode of operation may be associated with stabilizing the loop structure at the C-terminal end of the P6 helix of the P-domain, thereby affecting the function of highly conserved catalytic residues and promoting helix-helix interactions between the P- and A-domains in the E(2) state.     

Molecular architecture of the phosphorylation region of the yeast plasma membrane H+-ATPase

The molecular architecture of the yeast plasma membrane H(+)-ATPase phosphorylation region was explored by Fe(2+)-catalyzed cleavage. An ATP-Mg(2+).Fe(2+) complex was found to act as an affinity cleavage reagent in the presence of dithiothreitol/H(2)O(2). Selective enzyme cleavage required bound adenine nucleotide, either ATP or ADP, in the presence of Mg(2+). The fragment profile included a predominant N-terminal 61-kDa fragment, a minor 37-kDa fragment, and three prominent C-terminal fragments of 39, 36, and 30 kDa. The 61-kDa N-terminal and 39-kDa C-terminal fragments were predicted to originate from cleavage within the conserved MLT(558)GDAVG sequence. The 37-kDa fragment was consistent with cleavage within the S4/M4 sequence PVGLPA(340)V, while the 30-kDa and 36-kDa C-terminal fragments appeared to originate from cleavage in or around sequences D(646)TGIAVE and DMPGS(595)ELADF, respectively. The latter are spatially close to the highly conserved motif GD(634)GVND(638)APSL and conserved residues Thr(558) and Lys(615), which have been implicated in coordinating Mg(2+) and ATP. Overall, these results demonstrate that Fe(2+) associated with ATP and Mg(2+) acts as an affinity cleavage agent of the H(+)-ATPase with backbone cleavage occurring in conserved regions known to coordinate metal-nucleotide complexes. This study provides support for a three-dimensional organization of the phosphorylation region of the yeast plasma membrane H(+)-ATPase that is consistent with, but not identical to, typical P-type enzymes.     

Importance of Glu(282) in transmembrane segment M3 of the Na(+),K(+)-ATPase for control of cation interaction and conformational changes

Glu(282) located in the NH(2)-terminal part of transmembrane helix M3 of the Na(+),K(+)-ATPase was replaced by alanine, glycine, leucine, lysine, aspartate, or glutamine, and the effects of the mutations on the overall and partial reactions of the enzyme were analyzed. The mutations affected at least 3 important functions of the Na(+),K(+)-ATPase: (i) the conformational transitions between E(1) and E(2) forms of dephospho- and phosphoenzyme, (ii) Na(+) binding at the cytoplasmically facing sites of E(1), and (iii) long-range interaction controlling dephosphorylation. In mutants Glu(282) --> Lys and Glu(282) --> Asp, the E(1) form was favored during ATP hydrolysis, whereas the E(2) form was favored in Glu(282) --> Ala and Glu(282) --> Gly. Regardless of the change of conformational equilibrium, all the mutants displayed a reduced apparent affinity for Na(+), at least 3-fold for Glu(282) --> Lys and Glu(282) --> Asp, suggesting a direct effect on the Na(+) binding properties of E(1). Glu(282) --> Ala and Glu(282) --> Gly exhibited an extraordinary high rate of ATP hydrolysis in the mere presence of Na(+) without K(+) ("Na(+)-ATPase activity"), because of an increased rate of dephosphorylation of E(2)P. These results are in accordance with the hypothesis that Glu(282) is involved in the communication between the cation binding pocket and the catalytic site and in control of the cytoplasmic entry pathway for Na(+).     

Inhibition of phosphorylation of na+,k+-ATPase by mutations causing familial hemiplegic migraine

The neurological disorder familial hemiplegic migraine type II (FHM2) is caused by mutations in the α2-isoform of the Na(+),K(+)-ATPase. We have studied the partial reaction steps of the Na(+),K(+)-pump cycle in nine FHM2 mutants retaining overall activity at a level still compatible with cell growth. Although it is believed that the pathophysiology of FHM2 results from reduced extracellular K(+) clearance and/or changes in Na(+) gradient-dependent transport processes in neuroglia, a reduced affinity for K(+) or Na(+) is not a general finding with the FHM2 mutants. Six of the FHM2 mutations markedly affect the maximal rate of phosphorylation from ATP leading to inhibition by intracellular K(+), thereby likely compromising pump function under physiological conditions. In mutants R593W, V628M, and M731T, the defective phosphorylation is caused by local perturbations within the Rossmann fold, possibly interfering with the bending of the P-domain during phosphoryl transfer. In mutants V138A, T345A, and R834Q, long range effects reaching from as far away as the M2 transmembrane helix perturb the function of the catalytic site. Mutant E700K exhibits a reduced rate of E(2)P dephosphorylation without effect on phosphorylation from ATP. An extremely reduced vanadate affinity of this mutant indicates that the slow dephosphorylation reflects a destabilization of the phosphoryl transition state. This seems to be caused by insertion of the lysine between two other positively charged residues of the Rossmann fold. In mutants R202Q and T263M, effects on the A-domain structure are responsible for a reduced rate of the E(1)P to E(2)P transition.     

AS160 associates with the Na+,K+-ATPase and mediates the adenosine monophosphate-stimulated protein kinase-dependent regulation of sodium pump surface expression

The Na(+),K(+)-ATPase is the major active transport protein found in the plasma membranes of most epithelial cell types. The regulation of Na(+),K(+)-ATPase activity involves a variety of mechanisms, including regulated endocytosis and recycling. Our efforts to identify novel Na(+),K(+)-ATPase binding partners revealed a direct association between the Na(+),K(+)-ATPase and AS160, a Rab-GTPase-activating protein. In COS cells, coexpression of AS160 and Na(+),K(+)-ATPase led to the intracellular retention of the sodium pump. We find that AS160 interacts with the large cytoplasmic NP domain of the α-subunit of the Na(+),K(+)-ATPase. Inhibition of the activity of the adenosine monophosphate-stimulated protein kinase (AMPK) in Madin-Darby canine kidney cells through treatment with Compound C induces Na(+),K(+)-ATPase endocytosis. This effect of Compound C is prevented through the short hairpin RNA-mediated knockdown of AS160, demonstrating that AMPK and AS160 participate in a common pathway to modulate the cell surface expression of the Na(+),K(+)-ATPase.     

Arrestins and spinophilin competitively regulate Na+,K+-ATPase trafficking through association with a large cytoplasmic loop of the Na+,K+-ATPase

The activity and trafficking of the Na(+),K(+)-ATPase are regulated by several hormones, including dopamine, vasopressin, and adrenergic hormones through the action of G-protein-coupled receptors (GPCRs). Arrestins, GPCR kinases (GRKs), 14-3-3 proteins, and spinophilin interact with GPCRs and modulate the duration and magnitude of receptor signaling. We have found that arrestin 2 and 3, GRK 2 and 3, 14-3-3 epsilon, and spinophilin directly associate with the Na(+),K(+)-ATPase and that the associations with arrestins, GRKs, or 14-3-3 epsilon are blocked in the presence of spinophilin. In COS cells that overexpressed arrestin, the Na(+),K(+)-ATPase was redistributed to intracellular compartments. This effect was not seen in mock-transfected cells or in cells expressing spinophilin. Furthermore, expression of spinophilin appeared to slow, whereas overexpression of beta-arrestins accelerated internalization of the Na(+),K(+)-ATPase endocytosis. We also find that GRKs phosphorylate the Na(+),K(+)-ATPase in vitro on its large cytoplasmic loop. Taken together, it appears that association with arrestins, GRKs, 14-3-3 epsilon, and spinophilin may be important modulators of Na(+),K(+)-ATPase trafficking.     

NMR studies of the fifth transmembrane segment of Na+,K+-ATPase reveals a non-helical ion-binding region

The structure of a synthetic peptide corresponding to the fifth membrane-spanning segment (M5) in Na(+),K(+)-ATPase in sodium dodecyl sulfate (SDS) micelles was determined using liquid-state nuclear magnetic resonance (NMR) spectroscopy. The spectra reveal that this peptide is substantially less alpha-helical than the corresponding M5 peptide of Ca(2+)-ATPase. A well-defined alpha-helix is shown in the C-terminal half of the peptide. Apart from a short helical stretch at the N-terminus, the N-terminal half contains a non-helical region with two proline residues and sequence similarity to a non-structured transmembrane element of the Ca(2+)-ATPase. Furthermore, this region spans the residues implicated in Na(+) and K(+) transport, where they are likely to offer the flexibility needed to coordinate Na(+) as well as K(+) during active transport.     

Asymmetric orientation of amino groups in the alpha-subunit and the beta-subunit of (Na+ + K+)-ATPase in tight right-side-out vesicles of basolateral membranes from outer medulla

The orientation of amino groups in the membrane in the alpha- and beta-subunits of (Na+ + K+)-ATPase was examined by labeling with Boldon-Hunter reagent, N-succinimidyl 3-(4-hydroxy,5-[125I]iodophenyl)propionate), in right-side-out vesicles or in open membrane fragments from the thick ascending limbs of the Henles loop of pig kidney. Sealed right-side-out vesicles of basolateral membranes were separated from open membrane fragments by centrifugation in a linear metrizamide density gradient. After labeling, (Na+ + K+)-ATPase was purified using a micro-scale version of the ATP-SDS procedure. Distribution of label was analyzed after SDS-gel electrophoresis of alpha-subunit, beta-subunit and proteolytic fragments of alpha-subunit. Both the alpha- and the beta-subunit of (Na+ + K+)-ATPase are uniformly labeled, but the distribution of labeled residues on the two membrane surfaces differs markedly. All the labeled residues in the beta-subunit are located on the extracellular surface. In the alpha-subunit, 65-80% of modified groups are localized to the cytoplasmic surface and 20-35% to the extracellular membrane surface. Proteolytic cleavage provides evidence for the random distribution of 125I-labeling within the alpha-subunit. The preservation of (Na+ + K+)-ATPase activity and the observation of distinct proteolytic cleavage patterns of the E1- and E2-forms of the alpha-subunit show that the native enzyme structure is unaffected by labeling with Bolton-Hunter reagent. Bolton-Hunter reagent was shown not to permeate into sheep erythrocytes under the conditions of the labeling experiment. The data therefore allow the conclusion that the mass distribution is asymmetric, with all the labeled amino groups in the beta-subunit being on the extracellular surface, while the alpha-subunit exposes 2.6-fold more amino groups on the cytoplasmic than on the extracellular surface.