Involvement of the H3O+-Lys-164 -Gln-161-Glu-345 charge transfer pathway in proton transport of gastric H+,K+-ATPase

Gastric H(+),K(+)-ATPase is shown to transport 2 mol of H(+)/mol of ATP hydrolysis in isolated hog gastric vesicles. We studied whether the H(+) transport mechanism is due to charge transfer and/or transfer of hydronium ion (H(3)O(+)). From transport of [(18)O]H(2)O, 1.8 mol of water molecule/mol of ATP hydrolysis was found to be transported. We performed a molecular dynamics simulation of the three-dimensional structure model of the H(+),K(+)-ATPase alpha-subunit at E(1) conformation. It predicts the presence of a charge transfer pathway from hydronium ion in cytosolic medium to Glu-345 in cation binding site 2 (H(3)O(+)-Lys-164 -Gln-161-Glu-345). No charge transport pathway was formed in mutant Q161L, E345L, and E345D. Alternative pathways (H(3)O(+)-Gln-161-Glu-345) in mutant K164L and (H(3)O(+)-Arg-105-Gln-161-Gln-345) in mutant E345Q were formed. The H(+),K(+)-ATPase activity in these mutants reflected the presence and absence of charge transfer pathways. We also found charge transfer from sites 2 to 1 via a water wire and a charge transfer pathway (H(3)O(+)-Asn-794 -Glu-797). These results suggest that protons are charge-transferred from the cytosolic side to H(2)O in sites 2 and 1, the H(2)O comes from cytosolic medium, and H(3)O(+) in the sites are transported into lumen during the conformational transition from E(1)PtoE(2)P.     

Mutation of aspartate 804 of Na(+),K(+)-ATPase modifies the cation binding pocket and thereby generates a high Na(+)-ATPase activity

A series of six different mutants (D804A, D804E, D804G, D804N, D804Q, and D804S) of aspartate 804 present in transmembrane segment 6 of the rat Na(+),K(+)-ATPase alpha(1)-subunit were prepared and expressed in Sf9 cells by use of the baculovirus expression system. In contrast to the wild-type enzyme all mutants except D804Q showed a very high Na(+)-ATPase activity, which was hardly further stimulated by the addition of K(+). The ATPase activity of the mutants was already nearly maximal at 10 microM ATP and most of them could be phosphorylated in the absence of Na(+) at pH 6.0 and 21 degrees C, suggesting that they strongly prefer the E(1) over the E(2) conformation. However, Na(+) dose-dependently lowered the steady-state phosphorylation level, as a consequence of the increased affinity for Na(+) in the dephosphorylation reaction of the mutants compared to the wild-type enzyme. Conversely, the affinity for K(+) in the dephosphorylation reaction was decreased for the mutants as compared to that for the wild-type enzyme. When the pH was increased or the temperature was decreased, the phosphorylation level of the mutants decreased and the Na(+) activation in the phosphorylation reaction became apparent. It is concluded that upon mutation of aspartate 804 the affinity of the cation-binding pocket is changed relatively in favor of Na(+) instead of K(+), as a consequence of which the enzyme has obtained a preference for the E(1) conformation.     

Mimicking of K+ activation by double mutation of glutamate 795 and glutamate 820 of gastric H+,K+-ATPase

Six double mutants of Glu(795) and Glu(820) present in transmembrane domains 5 and 6 of the alpha-subunit of rat gastric H(+),K(+)-ATPase were generated and expressed with the baculovirus expression system. Five of the six mutants exhibited an SCH 28080-sensitive ATPase activity in the absence of K(+). The activity levels decreased in the following order: E795Q/E820A > E795Q/E820Q > E795Q/E820D congruent with E795A/E820A > E795L/E820Q. The E795L/E820D mutant possessed no constitutive activity. The relative low ATPase activity of the E795L/E820Q mutant is due to its low phosphorylation rate so that the dephosphorylation step was no longer rate-limiting. The constitutively active mutants showed a much lower vanadate sensitivity than the wild-type enzyme and K(+)-sensitive mutants, indicating that these mutants have a preference for the E(1) conformation. In contrast to the constitutively active single mutants generated previously, the double mutants exhibited a high spontaneous dephosphorylation rate at 0 degrees C compared to that of the wild-type enzyme. In addition, the H(+),K(+)-ATPase inhibitor SCH 28080 increased the steady-state phosphorylation level of the constitutively active mutants, due to the formation of a stable complex with the E(2)-P form. These studies further substantiate the idea that the empty ion binding pockets of some mutants apparently mimic the K(+)-filled binding pocket of the native enzyme.     

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.     

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(+).     

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.     

Alanine-scanning mutagenesis of the sixth transmembrane segment of gastric H+,K+-ATPase alpha-subunit

The sixth transmembrane (M6) segment of the catalytic subunit plays an important role in the ion recognition and transport in the type II P-type ATPase families. In this study, we singly mutated all amino acid residues in the M6 segment of gastric H(+),K(+)-ATPase alpha-subunit with alanine, expressed the mutants in HEK-293 cells, and studied the effects of the mutation on the functions of H(+),K(+)-ATPase; overall K(+)-stimulated ATPase, phosphorylation, and dephosphorylation. Four mutants, L819A, D826A, I827A, and L833A, completely lost the K(+)-ATPase activity. Mutant L819A was phosphorylated but hardly dephosphorylated in the presence of K(+), whereas mutants D826A, I827A, and L833A were not phosphorylated from ATP. We found that almost all of these amino acid residues, which are important for the function, are located on the same side of the alpha-helix of the M6 segment. In addition, we found that amino acids involved in the phosphorylation are located exclusively in the cytoplasmic half of the M6 segment and those involved in the K(+)-dependent dephosphorylation are in the luminal half. Several mutants such as I821A, L823A, T825A, and P829A partly retained the K(+)-ATPase activity accompanying the decrease in the rate of phosphorylation.     

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.     

The carbonyl group of glutamic acid-795 is essential for gastric H+,K+-ATPase activity

To study the role of Glu795offresent in the fifth transmembrane domain of the alpha-subunit of gastric H+,K+-ATPase, several mutants were generated and expressed in Sf9 insect cells. The E795Q mutant had rather similar properties as the wild-type enzyme. The apparent affinity for K+ in both the ATPase reaction and the dephosphorylation of the phosphorylated intermediate was even slightly enhanced. This indicates that the carbonyl group of Glu795 is sufficient for enzymatic activity. This carbonyl group, however, has to be at a particular position with respect to the other liganding groups, since the E795D and E795N mutants showed a strongly reduced ATPase activity, a lowered apparent K+ affinity, and a decreased steady-state phosphorylation level. In the absence of a carbonyl residue at position 795, the K+ sensitivity was either strongly decreased (E795A) or completely absent (E795L). The mutant E795L, however, showed a SCH 28080 sensitive ATPase activity in the absence of K+, as well as an enhanced spontaneous dephosphorylation rate, that could not be further enhanced by K+, suggesting that this mutant mimicks the filled K+ binding pocket. The results indicate that the Glu795 residue is involved in K+-stimulated ATPase activity and K+-induced dephosphorylation of the phosphorylated intermediate. Glu795 might also be involved in H+ binding during the phosphorylation step, since the mutants E795N, E795D, and E795A showed a decrease in the phosphorylation rate as well as in the apparent ATP affinity in the phosphorylation reaction. This indicates that Glu795 is not only involved in K+ but might also play a role in H+ binding.     

K(+)-independent gastric H(+),K(+)-atpase activity. Dissociation of K(+)-independent dephosphorylation and preference for the E1 conformation by combined mutagenesis of transmembrane glutamate residues

Several mutations of residues Glu(795) and Glu(820) present in M5 and M6 of the catalytic subunit of gastric H(+),K(+)-ATPase have resulted in a K(+)-independent, SCH 28080-sensitive ATPase activity, caused by a high spontaneous dephosphorylation rate. The mutants with this property also have a preference for the E(1) conformation. This paper investigates the question of whether these two phenomena are coupled. This possibility was studied by combining mutations in residue Glu(343), present in M4, with those in residues 795 and 820. When in combined mutants Glu and/or Gln residues were present at positions 343, 795, and 820, the residue at position 820 dominated the behavior: a Glu giving K(+)-activated ATPase activity and an E(2) preference and a Gln giving K(+)-independent ATPase activity and an E(1) preference. With an Asp at position 343, the enzyme could be phosphorylated, but the dephosphorylation was blocked, independent of the presence of either a Glu or a Gln at positions 795 and 820. However, in these mutants, the direction of the E(2) <--> E(1) equilibrium was still dominated by the 820 residue: a Glu giving E(2) and a Gln giving E(1). This indicates that the preference for the E(1) conformation of the E820Q mutation is independent of an active dephosphorylation process.     

Mutational effects on conformational changes of the dephospho- and phospho-forms of the Na+,K+-ATPase

Gly263 of the rat kidney Na(+),K(+)-ATPase is highly conserved within the family of P-type ATPases. Mutants in which Gly263 or the juxtaposed Arg264 had been replaced by alanine were expressed at high levels in COS-1 cells and characterized functionally. Titrations of Na(+),K(+), ATP, and vanadate dependencies of Na(+),K(+)-ATPase activity showed changes in the apparent affinities relative to wild-type compatible with a displacement of the E(1)-E(2) conformational equilibrium in favor of E(1). The level of the K(+)-occluded form was reduced in the Gly263-->Ala and Arg264-->Ala mutants, and the rate constant characterizing deocclusion of K(+) or Rb(+) was increased as much as 20-fold in the Gly263-->Ala mutant. Studies of the sensitivity of the phosphoenzyme to K(+) and ADP showed a displacement of the E(1)P-E(2)P equilibrium of the phosphoenzyme in favor of E(1)P, and dephosphorylation experiments carried out at 25 degrees C on a millisecond time scale using a quenched-flow technique demonstrated a reduction of the E(1)P to E(2)P conversion rate in the mutants. Hence, the mutations displaced the conformational equilibria of dephosphoenzyme and phosphoenzyme in parallel in favor of the E(1) and E(1)P forms. The observed effects were more pronounced in the Gly263-->Ala mutant compared with the Arg264-->Ala mutant. Leu332 mutations that likewise displaced the conformational equilibria in favor of E(1) and E(1)P were also studied. Unlike the Gly263-->Ala mutant the Leu332 mutants displayed a wild-type like rate of K(+) deocclusion. Thus, the effect of the Gly263 mutation on the E(1)-E(2) conformational equilibrium seems to be caused mainly by an acceleration of the K(+)-deoccluding step, whereas in the Leu332 mutants the rate of the reverse reaction seems to be reduced.     

The K(+) affinity of gastric H(+),K(+)-ATPase is affected by both lipid composition and the beta-subunit

It is generally assumed that negatively charged residues present in the alpha-subunit of gastric H(+),K(+)-ATPase are involved in K(+) binding and transport. Despite the fact that there is no difference between various species regarding these negatively charged residues, it was observed that the apparent K(+) affinity of the pig enzyme was much lower than that of the rat H(+),K(+)-ATPase. By determining the K(+)-stimulated dephosphorylation reaction of the phosphorylated intermediate K(0.5) values for K(+) of 0.12+/-0.01 and 1.73+/-0.03 mM were obtained (ratio 14.4) for the rat and the pig enzyme, respectively. To investigate the reason for the observed difference in K(+) sensitivity, both enzymes originating from the gastric mucosa were either reconstituted in a similar lipid environment or expressed in Sf9 cells. After reconstitution in K(+)-permeable phosphatidylcholine/cholesterol liposomes K(0.5) values for K(+) of 0.16+/-0.01 and 0.35+/-0.05 mM for the rat and pig enzyme respectively were measured (ratio 2.2). After expression in Sf9 cells the pig gastric H(+),K(+)-ATPase still showed a 4.1 times lower K(+) sensitivity than that of the rat enzyme. This means that the difference in K(+) sensitivity of the rat and pig gastric H(+), K(+)-ATPase is not only due to a different lipid composition but also to the structure of either the alpha- or beta-subunit. Expression of hybrid enzymes in Sf9 cells showed that the difference in K(+) sensitivity between the rat and pig gastric H(+),K(+)-ATPase is primarily due to differences in the beta-subunit.     

Halenaquinol, a natural cardioactive pentacyclic hydroquinone, interacts with sulfhydryls on rat brain Na(+),K(+)-ATPase

Halenaquinol inhibited the partial reactions of ATP hydrolysis by rat brain cortex Na(+),K(+)-ATPase, such as [3H]ATP binding to the enzyme, Na(+)-dependent front-door phosphorylation from [gamma-(33)P]ATP, and also Na(+)- and K(+)-dependent E(1)<-->E(2) conformational transitions of the enzyme. Halenaquinol abolished the positive cooperativity between the Na(+)- and K(+)-binding sites on the enzyme. ATP and sulfhydryl-containing reagents (cysteine and dithiothreitol) protected the Na(+),K(+)-ATPase against inhibition. Halenaquinol can react with additional vital groups in the enzyme after blockage of certain sulfhydryl groups with 5,5'-dithio-bis-nitrobenzoic acid. Halenaquinol inhibited [3H]ouabain binding to Na(+),K(+)-ATPase under phosphorylating and non-phosphorylating conditions. Binding of fluorescein 5'-isothiocyanate to Na(+),K(+)-ATPase and intensity of fluorescence of enzyme tryptophanyl residues were decreased by halenaquinol. We suggest that interaction of halenaquinol with the essential sulfhydryls in/or near the ATP-binding site of Na(+),K(+)-ATPase resulted in a change of protein conformation and subsequent alteration of overall and partial enzymatic reactions.     

Mutational study on the roles of disulfide bonds in the beta-subunit of gastric H+,K+-ATPase

The gastric proton pump, H(+),K(+)-ATPase, consists of the catalytic alpha-subunit and the non-catalytic beta-subunit. Correct assembly between the alpha- and beta-subunits is essential for the functional expression of H(+),K(+)-ATPase. The beta-subunit contains nine conserved cysteine residues; two are in the cytoplasmic domain, one in the transmembrane domain, and six in the ectodomain. The six cysteine residues in the ectodomain form three disulfide bonds. In this study, we replaced each of the cysteine residues of the beta-subunit with serine individually and in several combinations. The mutant beta-subunits were co-expressed with the alpha-subunit in human embryonic kidney 293 cells, and the role of each cysteine residue or disulfide bond in the alpha/beta assembly, stability, and cell surface delivery of the alpha- and beta-subunits and H(+),K(+)-ATPase activity was studied. Mutant beta-subunits with a replacement of the cytoplasmic and transmembrane cysteines preserved H(+),K(+)-ATPase activity. All the mutant beta-subunits with replacement(s) of the extracellular cysteines did not assemble with the alpha-subunit, resulting in loss of H(+),K(+)-ATPase activity. These mutants did not permit delivery of the alpha-subunit to the cell surface. Therefore, each of these disulfide bonds of the beta-subunit is essential for assembly with the alpha-subunit and expression of H(+),K(+)-ATPase activity as well as for cell surface delivery of the alpha-subunit.     

Reversible inhibitions of gastric H+,K(+)-ATPase by scopadulcic acid B and diacetyl scopadol. New biochemical tools of H+,K(+)-ATPase

Scopadulcic acid B (SA-B), a novel diterpenoid, is a main ingredient of the Paraguayan traditional medicinal herb "Typychá kuratú (Scoparia dulcis L.). SA-B and its debenzoyl derivative, diacetyl scopadol (DAS), specifically inhibit ATP hydrolysis of gastric H+,K(+)-ATPase. Both compounds inhibit the K(+)-dependent dephosphorylation step of the enzyme without any effect on the phosphorylation step. SA-B is a mixed-type inhibitor with respect to the activating cation, K+. SA-B lowers the affinity of H+,K(+)-ATPase to K+ and decreases the maximal velocity of ATP hydrolysis, whereas DAS is an uncompetitive inhibitor with respect to K+. Furthermore, the effects of SA-B and DAS on conformational states of the ATPase were studied by measuring the changes in the fluorescence intensity of the fluorescein isothiocyanate-labeled enzyme. The fluorescence study shows that SA-B primarily binds to the E2K form in the presence of Mg2+ and stabilizes the form and that DAS stabilizes the E2PK form. Therefore, the chemical modification of SA-B, debenzoylation, induced the changes in the pattern of inhibition of H+,K(+)-ATPase. Furthermore, the inhibition mechanisms of SA-B and DAS were different from those of omeprazole, which is an irreversible inhibitor, and SCH 28080, which is a reversible, competitive inhibitor with respect to K+. DAS also inhibited the K(+)-dependent p-nitrophenyl phosphatase activity, and the inhibition was competitive with respect to K+, indicating that the K(+)-dependent p-nitrophenylphosphatase activity does not represent the partial reaction step of H+,K(+)-ATPase.     

Translocation of Na(+),K(+)-ATPase is induced by Rho small GTPase in renal epithelial cells

The distribution of transmembrane proteins is considered to be crucial for their activities because these proteins mediate the information coming from outside of cells. A small GTPase Rho participates in many cellular functions through its downstream effectors. In this study, we examined the effects of RhoA on the distribution of Na(+),K(+)-ATPase, one of the transmembrane proteins. In polarized renal epithelium, Na(+),K(+)-ATPase is known to be localized at the basolateral membrane. By microinjection of the constitutively active mutant of RhoA (RhoA(Val14)) into cultured renal epithelial cells, Na(+),K(+)-ATPase was translocated to the spike-like protrusions over the apical surfaces. Microinjection of the constitutively active mutant of other Rho family GTPases, Rac1 or Cdcd42, did not induce the translocation. The translocation induced by RhoA(Val14) was inhibited by treatment with Y-27632, a Rho-kinase specific inhibitor, or by coinjection of the dominant negative mutant of Rho-kinase. These results indicate that Rho and Rho-kinase are involved in the regulation of the localization of Na(+),K(+)-ATPase. We also found that Na(+),K(+)-ATPase seemed to be colocalized with ERM proteins phosphorylated at T567 (ezrin), T564 (radixin), and T558 (moesin) in cells microinjected with RhoA(Val14).     

Evidence for tryptophan residues in the cation transport path of the Na(+),K(+)-ATPase

A family of aryl isothiouronium derivatives was designed as probes for cation binding sites of Na(+),K(+)-ATPase. Previous work showed that 1-bromo-2,4,6-tris(methylisothiouronium)benzene (Br-TITU) acts as a competitive blocker of Na(+) or K(+) occlusion. In addition to a high-affinity cytoplasmic site (K(D) < 1 microM), a low-affinity site (K(D) approximately 10 microM) was detected, presumably extracellular. Here we describe properties of Br-TITU as a blocker at the extracellular surface. In human red blood cells Br-TITU inhibits ouabain-sensitive Na(+) transport (K(D) approximately 30 microM) in a manner antagonistic with respect to extracellular Na(+). In addition, Br-TITU impairs K(+)-stimulated dephosphorylation and Rb(+) occlusion from phosphorylated enzyme of renal Na(+),K(+)-ATPase, consistent with binding to an extracellular site. Incubation of renal Na(+),K(+)-ATPase with Br-TITU at pH 9 irreversibly inactivates Na(+),K(+)-ATPase activity and Rb(+) occlusion. Rb(+) or Na(+) ions protect. Preincubation of Br-TITU with red cells in a K(+)-free medium at pH 9 irreversibly inactivates ouabain-sensitive (22)Na(+) efflux, showing that inactivation occurs at an extracellular site. K(+), Cs(+), and Li(+) ions protect against this effect, but the apparent affinity for K(+), Cs(+), or Li(+) is similar (K(D) approximately 5 mM) despite their different affinities for external activation of the Na(+) pump. Br-TITU quenches tryptophan fluorescence of renal Na(+),K(+)-ATPase or of digested "19 kDa membranes". After incubation at pH 9 irreversible loss of tryptophan fluorescence is observed and Rb(+) or Na(+) ions protect. The Br-TITU appears to interact strongly with tryptophan residue(s) within the lipid or at the extracellular membrane-water interface and interfere with cation occlusion and Na(+),K(+)-ATPase activity.     

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.     

Functional consequences of alterations to Pro328 and Leu332 located in the 4th transmembrane segment of the alpha-subunit of the rat kidney Na+,K(+)-ATPase.

Site-specific mutagenesis was used to analyse the functional roles of the residues Pro328 and Leu332 located in the conserved PEGLL motif of the predicted transmembrane helix M4 in the alpha 1-subunit of the ouabain resistant rat kidney Na+,K(+)-ATPase. cDNAs encoding either of the Na+,K(+)-ATPase mutants Pro328-->Ala and Leu332-->Ala, and wild type, were cloned into the expression vector pMT2 and transfected into COS-1 cells. Ouabain-resistant clones growing in the presence of 10 microM ouabain were isolated, and the Na+,K+, ATP and pH dependencies of the Na+,K(+)-ATPase activity measured in the presence of 10 microM ouabain were analysed. Under these conditions the exogenous expressed Na+,K(+)-ATPase contributed more than 95% of the Na+,K(+)-ATPase activity. The Pro328-->Ala mutant displayed a reduced apparent affinity for Na+ (K0.5 (Na+) 13.04 mM), relative to the wild type (K0.5 (Na+) 7.13 mM). By contrast, the apparent affinity for Na+ displayed by the Leu332-->Ala mutant was increased (K0.5 (Na+) 3.92 mM). Either of the mutants exhibited lower apparent affinity for K+ relative to the wild type (K0.5 (K+) 2.46 mM for Pro328-->Ala and 1.97 mM for Leu332-->Ala, compared with 0.78 mM for wild type). Both mutants exhibited higher apparent affinity for ATP than the wild type (K0.5 (ATP) 0.086 mM for Pro328-->Ala and 0.042 mM for Leu332-->Ala, compared with 0.287 mM for wild type). The influence of pH was in accordance with an acceleration of the E2 (K)-->E1 transition in the mutants relative to the wild type. These data are consistent with a role of Pro328 and Leu332 in the stabilization of the E2 form and of Pro328 in Na+ binding. The possible role of the mutated residues in K+ binding is discussed.     

Simultaneous phosphorylation of Ser11 and Ser18 in the alpha-subunit promotes the recruitment of Na(+),K(+)-ATPase molecules to the plasma membrane

Renal sodium homeostasis is a major determinant of blood pressure and is regulated by several natriuretic and antinatriuretic hormones. These hormones, acting through intracellular second messengers, either activate or inhibit proximal tubule Na(+),K(+)-ATPase. We have shown previously that phorbol ester (PMA) stimulation of endogenous PKC leads to activation of Na(+),K(+)-ATPase in cultured proximal tubule cells (OK cells) expressing the rodent Na(+), K(+)-ATPase alpha-subunit. We have now demonstrated that the treatment with PMA leads to an increased amount of Na(+),K(+)-ATPase molecules in the plasmalemma, which is proportional to the increased enzyme activity. Colchicine, dinitrophenol, and potassium cyanide prevented the PMA-dependent stimulation of activity without affecting the increased level of phosphorylation of the Na(+), K(+)-ATPase alpha-subunit. This suggests that phosphorylation does not directly stimulate Na(+),K(+)-ATPase activity; instead, phosphorylation may be the triggering mechanism for recruitment of Na(+),K(+)-ATPase molecules to the plasma membrane. Transfected cells expressing either an S11A or S18A mutant had the same basal Na(+),K(+)-ATPase activity as cells expressing the wild-type rodent alpha-subunit, but PMA stimulation of Na(+),K(+)-ATPase activity was completely abolished in either mutant. PMA treatment led to phosphorylation of the alpha-subunit by stimulation of PKC-beta, and the extent of this phosphorylation was greatly reduced in the S11A and S18A mutants. These results indicate that both Ser11 and Ser18 of the alpha-subunit are essential for PMA stimulation of Na(+), K(+)-ATPase activity, and that these amino acids are phosphorylated during this process. The results presented here support the hypothesis that PMA regulation of Na(+),K(+)-ATPase is the result of an increased number of Na(+),K(+)-ATPase molecules in the plasma membrane.