General and specific lipid-protein interactions in Na,K-ATPase.

The molecular activity of Na,K-ATPase and other P2 ATPases like Ca(2+)-ATPase is influenced by the lipid environment via both general (physical) and specific (chemical) interactions. Whereas the general effects of bilayer structure on membrane protein function are fairly well described and understood, the importance of the specific interactions has only been realized within the last decade due particularly to the growing field of membrane protein crystallization, which has shed new light on the molecular details of specific lipid-protein interactions. It is a remarkable observation that specific lipid-protein interactions seem to be evolutionarily conserved, and conformations of specifically bound lipids at the lipid-protein surface within the membrane are similar in crystal structures determined with different techniques and sources of the protein, despite the rather weak lipid-protein interaction energy. Studies of purified detergent-soluble recombinant αß or αßFXYD Na,K-ATPase complexes reveal three separate functional effects of phospholipids and cholesterol with characteristic structural selectivity. The observations suggest that these three effects are exerted at separate binding sites for phophatidylserine/cholesterol (stabilizing), polyunsaturated phosphatidylethanolamine (stimulatory), and saturated PC or sphingomyelin/cholesterol (inhibitory), which may be located within three lipid-binding pockets identified in recent crystal structures of Na,K-ATPase. The findings point to a central role of direct and specific interactions of different phospholipids and cholesterol in determining both stability and molecular activity of Na,K-ATPase and possible implications for physiological regulation by membrane lipid composition. This article is part of a special issue titled "Lipid-Protein Interactions."

Interaction of an aromatic dibromoisothiouronium derivative with the Ca(2+)-ATPase of skeletal muscle sarcoplasmic reticulum.

Isothiouronium compounds [Hoving, S., Bar-Shimon, M., Tijmes, J. J., Goldshleger, R., Tal, D. M., and Karlish, S. J. (1995) J. Biol. Chem. 270, 29788-29793] act as high-affinity competitive antagonists for Na(+) and K(+) (Rb(+)) on the renal Na(+)/K(+)-ATPase where they favor the E1 conformation. We have now characterized the effects of 1,3-dibromo-2,4,6-tris(methylisothiouronium)benzene (Br(2)-TITU) on the Ca(2+)-ATPase of skeletal muscle sarcoplasmic reticulum. Br(2)-TITU inhibited the Ca(2+)-ATPase, both transport and catalytic activity, with a K(0.5) of 5-15 microM. Maximum inhibition was at 10 min with t(0.5) of 3-5 min. Br(2)-TITU, 100 microM, quenched Trp autofluorescence by 80%, but the residual signal still responded to Ca(2+) binding. Maximum quenching of fluorescence was at pH 9.0. Total E-P levels, during the steady state of turnover of the Ca(2+)-ATPase, were increased from 0.5 to 5.8 nmol x mg(-1) by Br(2)-TITU at pH 6.8. Trinitrophenyl-ATP (TNP-ATP) superfluorescence, which monitors hydrophobicity of the ATP site, was increased 3-4-fold, suggesting that Br(2)-TITU favors an "E2"-like state. Fluorescence was also increased 3-5-fold when E-P was induced with P(i) plus EGTA. Br(2)-TITU increased the rate constants of induction of superfluorescence with ATP plus Ca(2+) from 0.32 to 0.69 s(-1) and with P(i) plus EGTA from 0.84 to 7.45 s(-1). Br(2)-TITU also decreased rate constants for "off" reactions from 2.9 to 0.66 s(-1) and from 10.9 to 0.73 s(-1) for the ATP and P(i) reactions, respectively. Br(2)-TITU, which competitively inhibits the Na(+)/K(+)-ATPase, has a novel effect on the Ca(2+)-ATPase. It promotes accumulation of E2-P species due to increased rate of formation and decreased rate of hydrolysis and quenches tryptophan autofluorescence. Br(2)-TITU could be a useful inhibitor to probe intermediate reactions of the Ca(2+)-ATPase that link catalysis with Ca(2+) translocation.

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.

Proximity of transmembrane segments M3 and M1 of the alpha subunit of Na+,K+-ATPase revealed by specific oxidative cleavage mediated by a complex of Cu2+ ions and 4,7-diphenyl-1,10-phenanthroline.

This paper describes a novel approach to specific oxidative cleavage of Na(+),K(+)-ATPase, mediated by Cu(2+) ions and a hydrophobic phenanthroline, 4,7-diphenyl-1,10-phenanthroline (DPP), in the presence of ascorbate and H(2)O(2). The cleavage produces two major fragments of the alpha subunit, with apparent molecular masses of 96.5 and 76 kDa, and N-termini near the cytoplasmic entrance of transmembrane segments M1 and M3, respectively, The kinetics indicate that both cleavages are mediated by a single Cu(2+)-DPP complex. We infer that M3 and M1 are in proximity near the cytoplasmic surface. The yields of 96.5 and 76 kDa fragments are not significantly affected by ligands that stabilize different E(1) and E(2) conformations. In E(2)(K) and E(2)P conformations, a minor 5.5 kDa fragment with its N-terminus in M10 is also observed. The 96.5 and 76 kDa fragments are indistinguishable from two fragments near M3 and M1 produced by Fe(2+)-catalyzed cleavage described previously [Goldshleger, R., and Karlish, S. J. D. (1999) J. Biol. Chem. 274, 16213-16221], whereas other Fe(2+)-catalyzed cleavage fragments in the cytoplasmic P and A domains are not observed with the Cu(2+)-DPP complex. These findings provide experimental support for the concept of two separate Fe(2+) sites. A homology model, with Na(+),K(+)-ATPase residues within transmembrane segments and connecting loops substituted into the crystal structure of Ca(2+)-ATPase, shows the proximity between the sequences HFIH in M3 and EVWK in M1, near the cytoplasmic surface. Thus, the model strongly supports the conclusions based on cleavages mediated by the Cu(2+)-DPP complex (or Fe(2+) at site 2). As a corollary, the cleavages provide evidence for similar packing of M1 and M3 of Na(+),K(+)-ATPase and Ca(2+)-ATPase.

Mutational analysis of the K+-competitive inhibitor site of gastric H,K-ATPase.

The gastric H,K-ATPase is inhibited selectively and K(+)-competitively from its luminal surface by protonated imidazo[1,2alpha]pyridines (e.g., SCH28080). Identification of the amino acids in the membrane domain that affect SCH28080 inhibition should provide a template for modeling a luminally directed vestibule in this enzyme, based on the crystal structure of the sr Ca-ATPase. Five conserved carboxylic residues, Glu343, Glu795, Glu820, Asp824, Glu936, and unique Lys791 in the H,K-ATPase were mutated, and the effects of mutations on the K(i) for SCH28080, V(max), and K(m,app)[NH(4)(+)] were measured. A kinetic analysis of the ATP hydrolysis data indicated that all of these residues significantly affect the interaction of NH(4)(+) ions with the protein but only three of them, Glu795, Glu936, and Lys791, greatly affected SCH28080 inhibition. A Glu795Asp mutation increased the K(i) from 64 +/- 11 to 700 +/- 110 nM. Since, however, the mutation Glu795Gln did not change the K(i) (86 +/- 31 nM), this site has a significant spatial effect on inhibitor kinetics. A Glu936Asp mutation resulted in noncompetitive kinetics while Gln substitution had no effect either on inhibitor affinity or on the nature of the kinetics, suggesting that the length of the Glu936 side chain is critical for the exclusive binding of the ion and SCH28080. Mutation of Lys791 to Ser, the residue present in the SCH28080-insensitive Na,K-ATPase, resulted in a 20-fold decrease in SCH28080 affinity, suggesting an important role of this residue in SCH28080 selectivity of the H,K-ATPase versus Na,K-ATPase. Mutations of Asp824, Glu343, and Glu820 increased the K(i) 2-3-fold, implying a relatively minor role for these residues in SCH28080 inhibition. It appears that the imidazopyridine moiety of SCH28080 in the protonated state interacts with residues near the negatively charged residues of the empty ion site from the luminal side (TM4, -5, -6, and -8) while the hydrophobic phenyl ring interacts with TM1 or TM2 (the latter conclusion based on previous data from photoaffinity labeling). The integrity of the SCH28080 binding site depends on the presence of Lys791, Glu936, and Glu795 in H,K-ATPase. A computer-generated model of this region illustrates the possible involvement of the residues previously shown to affect SCH28080 inhibition (Cys813, Ile816, Thr823, Met334, Val337) and may predict other residues that line the SCH28080 binding vestibule in the E(2) conformation of the pump.

Functional properties of Na,K-ATPase, and their structural implications, as detected with biophysical techniques.

A full understanding of the molecular mechanism of ion transport and energetics of the Na,K-ATPase will require both structural and functional data. During recent years biophysical methods have provided a number of important pieces of information on ion binding and release and the charge transfer process. This allows the formulation of kinetic models of the transport process. Although a breakthrough has not been obtained due to the lack of detailed knowledge on the three-dimensional structure with a resolution high enough to identify the ion-binding sites and the transport pathway, the functional information has structural implications that create constraints on possible mechanisms of active transport. Here we describe briefly contributions of some biophysical methods to our conceptual understanding of the ion transport process.

Functional role and immunocytochemical localization of the gamma a and gamma b forms of the Na,K-ATPase gamma subunit.

The gamma subunit of the Na,K-ATPase is a member of the FXYD family of type 2 transmembrane proteins that probably function as regulators of ion transport. Rat gamma is present primarily in the kidney as two main splice variants, gamma(a) and gamma(b), which differ only at their extracellular N termini (TELSANH and MDRWYL, respectively; Kuster, B., Shainskaya, A., Pu, H. X., Goldshleger, R., Blostein, R., Mann, M., and Karlish, S. J. D. (2000) J. Biol. Chem. 275, 18441-18446). Expression in cultured cells indicates that both variants affect catalytic properties, without a detectable difference between gamma(a) and gamma(b). At least two singular effects are seen, irrespective of whether the variants are expressed in HeLa or rat alpha1-transfected HeLa cells, i.e. (i) an increase in apparent affinity for ATP, probably secondary to a left shift in E(1) <--> E(2) conformational equilibrium and (ii) an increase in K(+) antagonism of cytoplasmic Na(+) activation. Antibodies against the C terminus common to both variants (anti-gamma) abrogate the first effect but not the second. In contrast, gamma(a) and gamma(b) show differences in their localization along the kidney tubule. Using anti-gamma (C-terminal) and antibodies to the rat alpha subunit as well as antibodies to identify cell types, double immunofluorescence showed gamma in the basolateral membrane of several tubular segments. Highest expression is in the medullary portion of the thick ascending limb (TAL), which contains both gamma(a) and gamma(b). In fact, TAL is the only positive tubular segment in the medulla. In the cortex, most tubules express gamma but at lower levels. Antibodies specific for gamma(a) and gamma(b) showed differences in their cortical location; gamma(a) is specific for cells in the macula densa and principal cells of the cortical collecting duct but not cortical TAL. In contrast, gamma(b) but not gamma(a) is present in the cortical TAL only. Thus, the importance of gamma(a) and gamma(b) may be related to their partially overlapping but distinct expression patterns and tissue-specific functions of the pump that these serve.

Structural organization and energy transduction mechanism of Na+,K+-ATPase studied with transition metal-catalyzed oxidative cleavage.

This chapter describes contributions of transition metal-catalyzed oxidative cleavage of Na+,K+-ATPase to our understanding of structure-function relations. In the presence of ascorbate/H2O2, specific cleavages are catalyzed by the bound metal and because more than one peptide bond close to the metal can be cleaved, this technique reveals proximity of the different cleavage positions within the native structure. Specific cleavages are catalyzed by Fe2+ bound at the cytoplasmic surface or by complexes of ATP-Fe2+, which directs the Fe2+ to the normal ATP-Mg2+ site. Fe2+- and ATP-Fe2+-catalyzed cleavages reveal large conformation-dependent changes in interactions between cytoplasmic domains, involving conserved cytoplasmic sequences, and a change of ligation of Mg2+ ions between E1P and E2P, which may be crucial in facilitating hydrolysis of E2P. The pattern of domain interactions in E1 and E2 conformations, and role of Mg2+ ions, may be common to all P-type pumps. Specific cleavages can also be catalyzed by Cu2+ ions, bound at the extracellular surfaces, or a hydrophobic Cu2+-diphenyl phenanthroline (DPP) complex, which directs the Cu2+ to the membrane-water interface. Cu2+ or Cu2+-DPP-catalyzed cleavages are providing information on alpha/beta subunit interactions and spatial organization of transmembrane segments. Transition metal-catalyzed cleavage could be widely used to investigate other P-type pumps and membrane proteins and, especially, ATP binding proteins.

Molecular and functional studies of the gamma subunit of the sodium pump.

This article reviews our studies of the gamma subunit of the sodium pump. Gamma is a member of the FXYD family of small, single transmembrane proteins and is expressed predominantly in the kidney tubule. There are two major variants of gamma which function similarly to bring about two distinct effects, one on K'(ATP) and the other, on K(K), the affinity of the pump for K+ acting as a competitor of cytoplasmic Na+. In this way, gamma is believed to provide a self-regulatory mechanism for maintaining the steady-state activity of the pump in the kidney. Our studies also suggest that K+ antagonism of cytoplasmic Na+ activation of the pump is relevant not only to the presence of gamma in the kidney, but probably some hitherto undefined factor(s) in other tissues, most notably heart. The interesting possibility that not only gamma but other members of the FXYD family regulate ion transport in a tissue-specific manner is discussed.

The complex ATP-Fe(2+) serves as a specific affinity cleavage reagent in ATP-Mg(2+) sites of Na,K-ATPase: altered ligation of Fe(2+) (Mg(2+)) ions accompanies the E(1)-->E(2) conformational change.

In the presence of ascorbate/H(2)O(2), ATP-Fe(2+) or AMP-PNP-Fe(2+) complexes act as affinity cleavage reagents, mediating selective cleavage of the alpha subunit of Na,K-ATPase at high affinity ATP-Mg(2+) sites. The cleavages reveal contact points of Fe(2+) or Mg(2+) ions. In E(1) and E(1)Na conformations, two major cleavages are detected within the conserved (708)TGDGVNDSPALKK sequence (at V712 and nearby), and one (E(1)Na) or two (E(1)) minor cleavages near V440. In media containing sodium and ATP, Fe(2+) substitutes for Mg(2+) in activating phosphorylation and ATP hydrolysis. In the E(1)P conformation, cleavages are the same as in E(1). Fe(2+) is not bound tightly. By contrast, in the E(2)P conformation, the pattern is different. A major cleavage occurs near the conserved sequence (212)TGES, whereas those in TGDGVNDSPALKK are less prominent. Fe(2+) is bound very tightly. On E(2)P hydrolysis, the Fe(2+) dissociates. The results are consistent with E(1)<-->E(2) conformation-dependent movements of cytoplasmic domains and sites for P(i) and Mg(2+) ions, inferred from previous Fe-cleavage experiments. Furthermore, these concepts fit well with the crystal structure of Ca-ATPase [Toyoshima, C., Nakasako, M., Nomura, H. & Ogawa, H. (2000) Nature (London) 405, 647-655]. Altered ligation of Mg(2+) ions in E(2)P may be crucial in facilitating nucleophilic attack of water on the OP bond. Mg(2+) ions may play a similar role in all P-type pumps. As affinity cleavage reagents, ATP-Fe(2+) or other nucleotide-Fe(2+) complexes could be widely used to investigate nucleotide binding proteins.

A new variant of the gamma subunit of renal Na,K-ATPase. Identification by mass spectrometry, antibody binding, and expression in cultured cells.

The gamma subunit is a specific regulator of Na,K-ATPase expressed mainly in kidney. On SDS-polyacryylamide gel electrophoresis, gamma runs as a doublet, but the origin and significance of the doublet is obscure. Mass spectrometry of the gamma chains of rat kidney Na, K-ATPase shows that gamma(a) (upper) has a mass of 7184.0 +/- 1 Da (carbamidomethyl cysteine), corresponding closely to that for the published sequence without the initiator methionine, while gamma(b) (lower) has a mass of 7337.9 +/- 1Da. Tryptic peptide mapping and sequencing by mass spectrometry reveals that the seven N-terminal residues of gamma(a), TELSANH, are replaced by Ac-MDRWYL in gamma(b), but otherwise the chains are identical. Antibodies raised against peptides TELSANHC and MDRWYLC recognize either gamma(a) or gamma(b) of the Na,K-ATPase, respectively. gamma(a) or gamma(b) cDNAs have been expressed in human embryonic kidney and HeLa cells. The major bands expressed correspond to gamma(a) or gamma(b) of renal Na, K-ATPase. Additional minor bands seen after transfection, namely gamma(a)' in human embryonic kidney and gamma(b)' in HeLa, are presumably cell-specific modifications. The present work clarifies earlier uncertainty regarding doublets seen in kidney and in transfected cells. In particular, the results show that renal Na, K-ATPase contains two variants of the gamma subunit with different sequences but otherwise are unmodified. We discuss the possible functional significance of the two variants.

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.

Evidence for an interaction between adducin and Na(+)-K(+)-ATPase: relation to genetic hypertension.

Adducin point mutations are associated with genetic hypertension in Milan hypertensive strain (MHS) rats and in humans. In transfected cells, adducin affects actin cytoskeleton organization and increases the Na(+)-K(+)-pump rate. The present study has investigated whether rat and human adducin polymorphisms differently modulate rat renal Na(+)-K(+)-ATPase in vitro. We report the following. 1) Both rat and human adducins stimulate Na(+)-K(+)-ATPase activity, with apparent affinity in tens of nanomolar concentrations. 2) MHS and Milan normotensive strain (MNS) adducins raise the apparent ATP affinity for Na(+)-K(+)-ATPase. 3) The mechanism of action of adducin appears to involve a selective acceleration of the rate of the conformational change E(2) (K) --> E(1) (Na) or E(2)(K). ATP --> E(1)Na. ATP. 4) Apparent affinities for mutant rat and human adducins are significantly higher than those for wild types. 5) Recombinant human alpha- and beta-adducins stimulate Na(+)-K(+)-ATPase activity, as do the COOH-terminal tails, and the mutant proteins display higher affinities than the wild types. 6) The cytoskeletal protein ankyrin, which is known to bind to Na(+)-K(+)-ATPase, also stimulates enzyme activity, whereas BSA is without effect; the effects of adducin and ankyrin when acting together are not additive. 7) Pig kidney medulla microsomes appear to contain endogenous adducin; in contrast with purified pig kidney Na(+)-K(+)-ATPase, which does not contain adducin, added adducin stimulates the Na(+)-K(+)-ATPase activity of microsomes only about one-half as much as that of purified Na(+)-K(+)-ATPase. Our findings strongly imply the existence of a direct and specific interaction between adducin and Na(+)-K(+)-ATPase in vitro and also suggest the possibility of such an interaction in intact renal membranes.

The energy transduction mechanism of Na,K-ATPase studied with iron-catalyzed oxidative cleavage.

This paper extends our recent report on specific iron-catalyzed oxidative cleavages of renal Na,K-ATPase and effects of E1 left arrow over right arrow E2 conformational transitions (Goldshleger, R. , and Karlish, S. J. D. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 9596-9601). The experiments indicate that only peptide bonds close to a bound Fe2+ ion are cleaved, and provide evidence on proximity of the different cleavage positions in the native enzyme. A sequence HFIH near trans-membrane segment M3 appears to be involved in Fe2+ binding. Previously we hypothesized that E2 and E1 conformations are characterized by formation or relaxation of interactions within the alpha subunit at or near highly conserved sequences, TGES in the minor cytoplasmic loop and CSDK, MVTGD, and VNDSPALKK in the major cytoplasmic loop. This concept has been tested by examining iron-catalyzed cleavage in both non-phosphorylated and phosphorylated conformations and effects of phosphate, vanadate, and ouabain. The results imply that both E1 left arrow over right arrow E2 and E1P left arrow over right arrow E2P transitions are indeed associated with formation and relaxation of interactions between cytoplasmic domains, comprising the minor loop plus N-terminal tail leading into M1 and major loop, respectively. Furthermore, it appears that either non-covalently or covalently bound phosphate bind near CSDK and MVTGD, and Mg2+ ions may bind to residues within TGES and VNDSPALKK and to bound phosphate. Thus cytoplasmic domain interactions seem to occur within or near the active site. We discuss the relationship between structural changes in the cytoplasmic domain and movements of trans-membrane segments that lead to cation transport. Presumably conformation-dependent formation and relaxation of domain interactions underlie energy transduction in all P-type pumps.

Expression and functional role of the gamma subunit of the Na, K-ATPase in mammalian cells.

The functional role of the gamma subunit of the Na,K-ATPase was studied using rat gamma cDNA-transfected HEK-293 cells and an antiserum (gammaC33) specific for gamma. Although the sequence for gamma was verified and shown to be larger (7237 Da) than first reported, it still comprises a single initiator methionine despite the expression of a gammaC33-reactive doublet on immunoblots. Kinetic analysis of the enzyme of transfected compared with control cells and of gammaC33-treated kidney pumps shows that gamma regulates the apparent affinity for ATP. Thus, gamma-transfected cells have a decreased K'ATP as shown in measurements of (i) K'ATP of Na,K-ATPase activity and (ii) K+ inhibition of Na-ATPase at 1 microM ATP. Consistent with the behavior of gamma-transfected cells, gammaC33 pretreatment increases K'ATP of the kidney enzyme and K+ inhibition (1 microM ATP) of both kidney and gamma-transfected cells. These results are consistent with previous findings that an antiserum raised against the pig gamma subunit stabilizes the E2(K) form of the enzyme (Therien, A. G., Goldshleger, R., Karlish, S. J., and Blostein, R. (1997) J. Biol. Chem. 272, 32628-32634). Overall, our data demonstrate that gamma is a tissue (kidney)-specific regulator of the Na,K-ATPase that can increase the apparent affinity of the enzyme for ATP in a manner that is reversible by anti-gamma antiserum.

Characterization of disulfide cross-links between fragments of proteolyzed Na,K-ATPase. Implications for spatial organization of trans-membrane helices.

This study characterizes disulfide cross-links between fragments of a well defined tryptic preparation of Na,K-ATPase, 19-kDa membranes solubilized with C12E10 in conditions preserving an intact complex of fragments and Rb occlusion (Or, E., Goldshleger, R., Tal, D. M., and Karlish, S. J. D. (1996) Biochemistry 35, 6853-6864). Upon solubilization, cross-links form spontaneously between the beta subunit, 19- and 11.7-kDa fragments of the alpha subunit, containing trans-membrane segments M7-M10 and M1/M2, respectively. Treatment with Cu2+-phenanthroline (CuP) improves efficiency of cross-linking. Sequencing and immunoblot analysis have shown that the cross-linked products consist of a mixture of beta-19 kDa dimers ( approximately 65%) and beta-19 kDa-11.7 kDa trimers ( approximately 35%). The alpha-beta cross-link has been located within the 19-kDa fragment to a 6.5-kDa chymotryptic fragment containing M8, indicating that betaCys44 is cross-linked to either Cys911 or Cys930. In addition, an internal cross-link between M9 and M10, Cys964-Cys983, has been found by sequencing tryptic fragments of the cross-linked product. The M1/M2-M7/M10 cross-link has not been identified directly. However, we propose that Cys983 in M10 is cross-linked either to Cys104 in M1 or internally to Cys964 in M9. Based on this study, cross-linking induced by o-phthalaldehyde (Or, E., Goldshleger, R., and Karlish, S. J. D. (1998) Biochemistry 37, 8197-8207), and information from the literature, we propose an approximate spatial organization of trans-membrane segments of the alpha and beta subunits.

Specific Cu2+-catalyzed oxidative cleavage of Na,K-ATPase at the extracellular surface.

This paper describes specific Cu2+-catalyzed oxidative cleavage of alpha and beta subunits of Na,K-ATPase at the extracellular surface. Incubation of right side-out renal microsomal vesicles with Cu2+ ions, ascorbate, and H2O2 produces two major cleavages of the alpha subunit within the extracellular loop between trans-membrane segments M7 and M8 and L7/8. Minor cleavages are also detected in loops L9/10 and L5/6. In the beta subunit two cleavages are detected, one before the first S-S bridge and the other between the second and third S-S bridges. Na,K-ATPase and Rb+ occlusion are inactivated after incubation with Cu2+/ascorbate/H2O2. These observations are suggestive of a site-specific mechanism involving cleavage of peptide bonds close to a bound Cu2+ ion. This mechanism allows several inferences on subunit interactions and spatial organization. The two cleavage sites in L7/8 of the alpha subunit and two cleavage sites of the beta subunit identify interacting segments of the subunits. L7/8 is also close to L9/10 and to cation occlusion sites. Comparison of the locations of Cu2+-catalyzed cleavages with Fe2+-catalyzed cleavages (Goldshleger, R., and Karlish, S. J. D. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 9596-9601) suggests division of the membrane sector into two domains comprising M1-M6 and M7-M10/Mbeta, respectively.

Metal-catalysed cleavage of Na,K-ATPase as a tool for study of structure-function relations.

This paper describes a novel technique for specific cleavage of renal Na/K-ATPase, based on bound transition metal ions. The approach might have application to other P-type pumps or membrane proteins. In one type of experiment, specific cleavages of the alpha subunit have been observed following incubation with ascorbate plus H2O2. Five fragments with intact C-terminals and complementary fragments with intact N-terminals are detectable. The beta subunit is not cleaved. Cleavages depend on the presence of contaminant or added submicromolar concentrations of Fe2+ ions. The results suggest that Fe2+ (or Fe3+) binds with high affinity at the cytoplasmic surface and catalyze cleavages of peptide bonds close to the Fe2+ (or Fe3+) ion. The rate of cleavage is greatly affected by the conformational state of the protein, E1Na or E2(Rb), respectively. The findings provide information on spatial organization of the protein and suggest that the highly conserved regions of the alpha subunit, within the minor and major cytoplasmic loops, interact in the E2 or E2(Rb) conformations, but move apart in the E1 or E1Na conformations. In a second application of this technique, added Cu2+ ions at micromolar concentrations, have been shown to catalyse specific cleavages of both alpha and beta subunits at the extracellular surface. The experiments provide evidence for trans-membrane topology and proximity between trans-membrane segments M5-M10 within the alpha subunit and for interacting segments of alpha and beta subunits. We discuss the implications of metal-catalysed cleavages for spatial organisation of transmembrane helices of the protein.

Conformational alterations resulting from mutations in cytoplasmic domains of the alpha subunit of the Na,K-ATPase.

This paper summarizes experiments concerned with the functional consequences of mutations in cytoplasmic regions of the alpha 1 subunit of the Na,K-ATPase, in particular the amino terminus, the first cytoplasmic loop between transmembrane segments M2 and M3, and the major cytoplasmic loop between M4 and M5. In the first mutation (alpha 1M32), 32 residues were removed from the N-terminus. The second mutation (E233K) was in the putative beta strand of M2-M3 loop and the third, comprised the replacement of the amino terminal half of loop M4-M5 of the Na,K-ATPase with the homologous segment (residues 356-519) of the gastric H,K-ATPase. The first two mutations, either separately or in combination (alpha 1M32E233K), shift the equilibrium between the major conformational states of the enzyme, E1 and E2, in favor of E1 as manifested by increased apparent affinity for ATP, lower catalytic turnover, and decreased sensitivity to inhibition by vanadate. The striking changes observed with alpha 1M32E233K suggests interactions between the N-terminus, the beta-strand in the M2-M3 loop and the catalytic phosphorylation site. The behavior of these mutants contrasts with that of least one mutant involving substitution of a residue in the putative cation binding pocket, namely S775A in the fifth transmembrane segment (Arguello, J.M., & Lingrel, J. B. J. Biol. Chem. 270: 22764-22771, 1995). Although its K+/ATP antagonism resembles that of the foregoing cytoplasmic mutants, its vanadate sensitivity is unaltered suggesting that changes in apparent affinity for ATP are secondary to changes in K+ ligation. The question of cation selectivity, in particular that of Na+ versus protons, has been addressed in structure/function analysis of a cytoplasmic chimera involving the M4-M5 loop. Transport studies performed in the presence or absence of Na+ and at low versus high pH indicate a marked alteration in cation affinity and/or selectivity. This results suggests coupling of an alteration in the large M4-M5 cytoplasmic domain to cation binding in, presumably, the juxtapositioned transmembrane domain.