Biomedical subjects

S J Karlish

Publications and source records attributed to S J Karlish.

At least 19 recordsLinked to original sources

PubMed2001

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.

Amino Acid Sequence↗

PubMed2001

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.

Amino Acid Sequence↗

PubMed2001

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.

Amino Acid Sequence↗

PubMed2001

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.

Adenosine Triphosphate↗

PubMed2001

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.

Protein Conformation↗

PubMed2000

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.

Adenosine Triphosphate↗

PubMed2000

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.

Amino Acid Sequence↗

PubMed2000

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.

Amino Acid Sequence↗

PubMed1999

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.

Animals↗

PubMed1999

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.

Amino Acid Sequence↗

PubMed1999

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.

Animals↗

PubMed1999

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.

Animals↗

PubMed1998

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.

Amino Acid Sequence↗

PubMed1998

Specific cross-links between fragments of proteolyzed Na,K-ATPase induced by o-phthalaldehyde.

We have used o-phthalaldehyde (OPA) to cross-link adjacent fragments of "19 kDa membranes", a tryptic preparation of Na,K-ATPase lacking the ATP site but retaining cation occlusion sites. Treatment with OPA of "19 kDa membranes" or detergent-solubilized membranes containing occluded Rb ions [Or, E., Goldshleger, R., Tal, D. M., and Karlish, S. J. D. (1996) Biochemistry 35, 6853-6864] yielded cross-linked products of 25 and 31 kDa. Both species contained the 19 kDa fragment of the alpha subunit (transmembrane segments M7-M10). In addition, the 25 kDa product contained the fragment including M5-M6, while the 31 kDa product contained a 16 kDa fragment of the beta subunit. Cross-linking was unaffected by the absence or presence of ligands (Na, Rb, or Mg and ouabain). Cross-linking was largely abolished in thermally inactivated "19 kDa membranes". When proteolytic digestion of the 25 and 31 kDa products was combined with antibody binding, PKA-dependent phosphorylation, and sequencing of fragments, approximate positions of the cross-links were established. In the 25 kDa product, the cross-link was located within the short cytoplasmic segment Asn831-Arg841 of the 19 kDa fragment preceding M7 and within Ala749-Ala770 preceding M5. Thus, M7 and M5 are likely to be in close proximity. In the 31 kDa product, the cross-link was located in the extracellular loop of the alpha subunit between M7 and M8, close to residues which are known to interact with the beta subunit. Functional implications of the interactions between the fragments of the alpha (M5-M6 and M7-M10) and beta subunits are discussed.

Animals↗

PubMed1998

Identification of the site of inhibition by omeprazole of a alpha-beta fusion protein of the H,K-ATPase using site-directed mutagenesis.

The alpha subunit of eukaryotic P-type ATPases has ten experimentally defined transmembrane or membrane inserted segments. The fifth and sixth of these are short, not predicted by hydropathy analysis, do not insert independently into microsomal membranes, and are readily removed after tryptic digestion and therefore may be membrane inserted sequences. Acid transport by the gastric H, K-ATPase is covalently inhibited by several substituted pyridyl methylsulfinyl benzimidazoles, such as omeprazole. These act as probes of accessible extracytoplasmic thiols because they are accumulated in the acid transporting gastric vesicles and then convert to thiol reactive, cationic tetracyclic sulfenamides. Inhibition is due mainly to disulfide formation with Cys813 or Cys822 in M5/6 and perhaps with a contribution from Cys892 in the loop between transmembrane segment (TM) 7 and TM8. Identification of the specific cysteine responsible for inhibition should be able to define the turn between M5 and M6. The gastric H,K-ATPase alpha-beta heterodimer was expressed as a fusion protein in HEK 293 cells. Transient transfection resulted in most of the protein being retained in the endoplasmic reticulum with only core glycosylation and minor activity of the ATPase evident. Stable transfection resulted in plasma membrane localization of the protein and complex glycosylation. The transfected but not the control cells displayed cation-stimulated, SCH 28080-inhibited ATPase activity and SCH 28080- and omeprazole-inhibited 86Rb uptake. The two cysteines in M5/6 and Cys892 in the TM7/8 loop were mutated to the amino acids found in the Na,K-ATPase in order to determine which of the three cysteine residues were important for benzimidazole inhibition. Mutation of one, two, or all three cysteines did not alter enzyme activity, 86Rb transport, or SCH 28080 inhibition. Only removal of Cys822 blocked omeprazole inhibition of 86Rb transport. These data suggest that Cys822 is present in a region of the enzyme most easily accessed by the cationic sulfenamide formed by omeprazole, presumably the turn between M5 and M6.

Amino Acid Sequence↗

PubMed1998

Interactions between fragments of trypsinized Na,K-ATPase detected by thermal inactivation of Rb+ occlusion and dissociation of the M5/M6 fragment.

This work provides evidence for interactions between fragments of "19-kDa membranes," a trypsinized preparation of Na,K-ATPase that retains cation occlusion and ouabain binding. Previously, we reported rapid thermal inactivation of Rb+ occlusion at 37 degreesC (Or, E., David, P., Shainskaya, A., Tal, D. M., and Karlish, S. J. D. (1993) J. Biol. Chem. 268, 16929-16937). We describe here the detailed kinetics of thermal inactivation. In the range 25-35 degreesC, a two-step model (N left and right arrow U --> I, where N is the native species, U is the reversibly unfolded intermediate, and I is the irreversibly denatured form) fits the data. Reversibility of inactivation has been observed at 25 degreesC, consistent with the model. At 37 degreesC and higher temperatures, the data can be fitted to the simple mechanism N --> I, i.e. U is not significant as an intermediate. Occluded cations (Na+, Rb+, K+, Tl+, NH4+, and Cs+) and ouabain protect strongly against thermal inactivation. Ca2+, Ba2+, and La3+ ions do not protect. Proteolysis experiments provide independent evidence that disorganization can occur in stages, first in transmembrane segments and then in extra-membrane segments of the fragments. Analysis of selective dissociation of the M5/M6 fragment at 37 degreesC (Lutsenko, S., and Kaplan, J. H. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 7936-7940), using a specific antibody, showed that inactivation of Rb+ occlusion precedes dissociation of the fragment, and only approximately 50% of the fragment is released when occlusion is fully inactivated. In the presence of Ca2+ ions, occlusion is inactivated, but the M5/M6 fragment is not released. The experiments demonstrate that occlusion is inactivated by disruption of interactions between fragments of 19-kDa membranes, and only then does the M5/M6 fragment dissociate. Interactions between the M5/M6 and M7/M10 fragments seem to be essential for maintenance of Rb+ occlusion.

Animals↗

PubMed1998

Importance of intramembrane carboxylic acids for occlusion of K+ ions at equilibrium in renal Na,K-ATPase.

Site-directed mutagenesis and assay of Rb+ and Tl+ occlusion in recombinant Na,K-ATPase from yeast were combined to establish structure-function relationships of amino acid side chains involved in high-affinity occlusion of K+ in the E2[2K] form. The wild-type yeast enzyme was capable of occluding 2 Rb+ or Tl+ ions/ouabain binding site or alpha 1 beta 1 unit with high apparent affinity (Kd(Tl+) = 7 +/- 2 microM), like the purified Na,K-ATPase from pig kidney. Mutations of Glu327(Gln,Asp), Asp804(Asn, Glu), Asp808(Asn, Glu) and Glu779(Asp) abolished high-affinity occlusion of Rb+ or Tl+ ions. The substitution of Glu779 for Gln reduced the occlusion capacity to 1 Tl+ ion/alpha 1 beta 1-unit with a 3-fold decrease of the apparent affinity for the ion (Kd(Tl+) = 24 +/- 8 microM). These effects on occlusion were closely correlated to effects of the mutations on K0.5(K+) for K+ displacement of ATP binding. Each of the four carboxylate residues Glu327, Glu779, and Asp804 or Asp808 in transmembrane segments 4, 5, and 6 is therefore essential for high-affinity occlusion of K+ in the E2[2K] form. These residues either may engage directly in cation coordination or they may be important for formation or stability of the occlusion cavity.

Adenosine Triphosphate↗

PubMed1998

Vanadyl ions stimulate K+ uptake into isolated perfused rat liver via the Na+/K+-pump by a tyrosine kinase-dependent mechanism.

Vanadium salts mimic most metabolic effects of insulin in vitro. We report here that vanadyl sulfate (VOSO4) and sodium vanadate (NaVO3) stimulate net K+ uptake in isolated perfused rat liver. Stimulation was evident at low concentrations of vanadyl ions (range 1-20 microM) and occurred within minutes following the addition of VOSO4. By comparison with VOSO4, insulin had less of a stimulatory effect on K+ uptake. Ouabain prevented the activating effect of VOSO4 on K+ uptake. Following a VOSO4 challenge, measured intracellular Na+ concentration ([Na+]i) fell (control, 17.1 +/- 1.2; VOSO4-treated, 13.0 +/- 1.1 mmol.g-1 wet weight, P = 0.027). The results indicate that active K+ uptake via the Na+/K+-ATPase was stimulated by vanadyl ions. An indirect mechanism due to changes in [Na+]i can be excluded. The tyrosine kinase inhibitor genistein was found to inhibit stimulation of K+ by vanadyl and vanadate ions which are known inhibitors of phosphotyrosine phosphatases. We conclude that stimulation of active K+ influx involves a tyrosine kinase. Possible mechanisms include phosphorylation at tyrosine residues and direct activation of the Na+/K+-ATPase, or phosphorylation of other proteins that regulate the activity or number of pumps in the cells.

Animals↗