Glu-857 moderates K+-dependent stimulation and SCH 28080-dependent inhibition of the gastric H,K-ATPase.

The rabbit H,K-ATPase alpha- and beta-subunits were transiently expressed in HEK293 T cells. The co-expression of the H,K-ATPase alpha- and beta-subunits was essential for the functional H,K-ATPase. The K+-stimulated H,K-ATPase activity of 0.82 +/- 0.2 micromol/mg/h saturated with a K0.5 (KCl) of 0.6 +/- 0.1 mM, whereas the 2-methyl-8-(phenylmethoxy)imidazo[1,2a]pyridine-3-acetonitrile (SCH 28080)-inhibited ATPase of 0.62 +/- 0.07 micromol/mg/h saturated with a Ki (SCH 28080) of 1.0 +/- 0.3 microM. Site mutations were introduced at the N,N-dicyclohexylcarbodiimide-reactive residue, Glu-857, to evaluate the role of this residue in ATPase function. Variations in the side chain size and charge of this residue did not inhibit the specific activity of the H,K-ATPase, but reversal of the side chain charge by substitution of Lys or Arg for Glu produced a reciprocal change in the sensitivity of the H,K-ATPase to K+ and SCH 28080. The K0.5 for K+stimulated ATPase was decreased to 0.2 +/-.05 and 0.2 +/-.03 mM, respectively, in Lys-857 and Arg-857 site mutants, whereas the Ki for SCH 28080-dependent inhibition was increased to 6.5 +/- 1.4 and 5.9 +/- 1.5 microM, respectively. The H,K-ATPase kinetics were unaffected by the introduction of Ala at this site, but Leu produced a modest reciprocal effect. These data indicate that Glu-857 is not an essential residue for cation-dependent activity but that the residue influences the kinetics of both K+ and SCH 28080-mediated functions. This finding suggests a possible role of this residue in the conformational equilibrium of the H,K-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.     

Interaction of a K(+)-competitive inhibitor, a substituted imidazo[1,2a] pyridine, with the phospho- and dephosphoenzyme forms of H+, K(+)-ATPase

Defining the structural and catalytic properties of the ion transport site(s) of enzyme-phosphorylating ATPases is of key importance in understanding the mechanism of ion transport by these enzymes. In the case of the H+, K(+)-ATPase, SCH 28080 (3-(cyanomethyl)-2-methyl-8-(phenylmethoxy)imidazo[1,2a]-pyridine) has been shown to act as a high affinity, extracytosolic, K(+)-competitive inhibitor of Mg2+, K(+)-ATPase activity (Wallmark, B., Briving, C., Fryklund, J., Munson, K., Jackson, R., Mendlein, J., Rabon, E., and Sachs, G. (1987) J. Biol. Chem. 262, 2077-2084). To define the nature of the SCH 28080-binding site in relation to the catalytic cycle of the enzyme, we have investigated the effects of this potential K+ transport site probe on the steady-state and partial reactions of the H+, K(+)-ATPase. In the absence of K+, SCH 28080 inhibits Mg2(+)-ATPase activity with high affinity (apparent Ki = 30 nM). Inhibition is due to K(+)-like prevention of phosphoenzyme formation. SCH 28080 has no effect on Mg2(+)-catalyzed dephosphorylation. SCH 28080, at concentrations less than 0.5 microM, increases the apparent Km for K+ for Mg2+, K(+)-ATPase activity with little effect on the maximum velocity. At higher concentrations of SCH 28080, reversal of inhibition by higher K+ concentrations is not complete, due to inhibition of ATPase activity by high K+. In contrast, SCH 28080 inhibits K(+)-stimulated dephosphorylation by competitively displacing K+ from phosphoenzyme with an extracytosolic conformation of the monovalent cation site (E2P) at low concentrations of SCH 28080 and K+. At higher concentrations, 10 microM SCH 28080 and 50 mM K+, a slowly dephosphorylating complex with both SCH 28080 and K+ bound to E2P may form which represents a small fraction of the total E2P (15-25%). Preincubation of SCH 28080 with E2P completely blocks K(+)-stimulated dephosphorylation, and K+ is unable to reverse this preincubation effect, indicating that the SCH 28080 dissociation rate is at least as slow as K(+)-independent dephosphorylation of E2P. These findings indicate that SCH 28080 inhibits K(+)-stimulated ATPase activity by competing with K+ for binding to E2P and blocking K(+)-stimulated dephosphorylation. In the absence of K+, SCH 28080 has a higher apparent affinity for E2P, but it permits K(+)-independent dephosphorylation. Since the dissociation rate of SCH 28080 from the enzyme is slow, phosphoenzyme formation is prevented by SCH 28080 remaining bound to the extracytosolic conformation of the monovalent cation site, thereby reducing the steady-state level of phosphoenzyme.     

The Cl- channel in hog gastric vesicles is part of the function of H,K-ATPase

Hog gastric vesicles showed Cl- conductance when treated with Cu2+-o-phenanthroline, an S-S cross-linking reagent. An IgG monoclonal antibody caused dose-dependent inhibition of Cl- conductance that had been induced by S-S cross-linking. The antibody did not cause intervesicular aggregation, as determined by measurement of vesicle size. These results show that Cl- conductance, the stimulation and inhibition of which are regulated reversibly by S-S----2SH transformation, is due to native, physiological channels. The antibody also dose dependently inhibited the activities of H,K-ATPase and p-nitrophenyl phosphatase in gastric vesicles, but did not inhibit Na,K-ATPase obtained from dog kidney. Immunoblotting with the antibody of vesicle proteins solubilized in sodium dodecyl sulfate-polyacrylamide gel showed that the antibody binds to a 95-kDa subunit of H,K-ATPase and its dimeric 180-kDa polypeptide. The antibody-binding sites of H,K-ATPase activity and the Cl- channel for the inhibition were present on the external (cytosolic) surface of the transmembraneous ATPase. A gastric antisecretory compound, 2-methyl-8-(phenylmethoxy)imidazo[1,2 alpha] pyridine-3-acetonitrile (SCH 28080), competitively bound to the high affinity site of K+ on the internal (luminal) surface of H,K-ATPase, and its half-maximal inhibitory concentration for H,K-ATPase activity in tight vesicles was 0.2 microM in the presence of valinomycin. SCH 28080 also dose dependently inhibited opening of Cl- channels by S-S cross-linking, the regulatory site being present on the cytosolic side and more internally than the antibody binding site. The half-inhibitory concentration of SCH 28080 was 0.3 microM. The present results with the antibody and SCH 28080 indicate that the Cl- channel is part of the function of H,K-ATPase.     

Comparison of covalent with reversible inhibitor binding sites of the gastric H,K-ATPase by site-directed mutagenesis

The gastric H,K-ATPase is covalently inhibited by substituted pyridyl-methylsulfinyl-benzimidazoles, such as omeprazole, that convert to thiophilic probes of luminally accessible cysteines in the acid space. The K(+) competitive inhibitor, SCH28080, prevented inhibition of acid transport by omeprazole. In stably expressing HEK293 cells, the benzimidazole-reactive cysteines, Cys-321 (transmembrane helix (TM) 3), Cys-813 and Cys-822 (TM5/6), and Cys-892 (TM7/8) were mutated to the amino acids found in the SCH28080-resistant Na,K-ATPase and kinetic parameters of H,K-ATPase activity analyzed. Mutations of Cys-822 and Cys-892 had insignificant effects on the K(i(app)), K(m(app)) or V(max), but mutations of Cys-813 to threonine and Cys-321 to alanine decreased the affinity for SCH28080. Mutation of Cys-321 to alanine produced mixed kinetics of inhibition, still with higher affinity for the cation-free form of phosphoenzyme. Since the phenylmethoxy ring of the imidazo-pyridine inhibitors binds to TM1/2, as shown by earlier photoaffinity studies, and the mutations in TM6 (Cys-813 --> Thr) as well as the end of TM3 (Cys-321 --> Ala) decrease the affinity for SCH28080, the TM1/2, TM3, and TM6 helices lie within approximately 16 A of each other based on the size of the active, extended conformation of SCH28080.     

Effects of mutations in M4 of the gastric H+,K+-ATPase on inhibition kinetics of SCH28080

The effects of site-directed mutagenesis were used to explore the role of residues in M4 on the apparent Ki of a selective, K+-competitive inhibitor of the gastric H+,K+ ATPase, SCH28080. A double transfection expression system is described, utilizing HEK293 cells and separate plasmids encoding the alpha and beta subunits of the H+,K+-ATPase. The wild-type enzyme gave specific activity (micromoles of Pi per hour per milligram of expressed H+,K+-ATPase protein), apparent Km for ammonium (a K+ surrogate), and apparent Ki for SCH28080 equal to the H+, K+-ATPase purified from hog gastric mucosa. Amino acids in the M4 transmembrane segment of the alpha subunit were selected from, and substituted with, the nonconserved residues in M4 of the Na+, K+-ATPase, which is insensitive to SCH28080. Most of the mutations produced competent enzyme with similar Km,app values for NH4+ and Ki,app for SCH28080. SCH28080 affinity was decreased 2-fold in M330V and 9-fold in both M334I and V337I without significant effect on Km,app. Hence methionine 334 and valine 337 participate in binding but are not part of the NH4+ site. Methionine 330 may be at the periphery of the inhibitor site, which must have minimum dimensions of approximately 16 x 8 x 5 A and be accessible from the lumen in the E2-P conformation. Multiple sequence alignments place the membrane surface near arginine 328, suggesting that the side chains of methionine 334 and valine 337, on one side of the M4 helix, project into a binding cavity within the membrane domain.     

Catalytic function of nongastric H,K-ATPase expressed in Sf-21 insect cells

We previously demonstrated that the alpha-subunit of human nongastric H,K-ATPase (Atp1al1) can assemble with the gastric H,K-ATPase beta-subunit (betaHK) into an active ion pump upon coexpression in Xenopus oocytes. To gain insight into enzymatic functions, we have analyzed the Atp1al1-betaHK complex using a baculovirus expression system. The efficient formation of the functional Atp1al1-betaHK complex in membranes of Sf-21 insect cells was obtained upon co-infection with recombinant baculoviruses expressing Atp1al1 and betaHK. Expression of either protein alone did not produce active ATPase. The effects of K(+), Na(+), pH, and ATP and inhibitors on ATPase activity of the recombinant Atp1al1-betaHK complex were analyzed. The Atp1al1-betaHK complex was shown to exhibit significant ATPase activity in nominally K(+)-free medium. The addition of K(+) stimulated the ATP hydrolysis up to 3-fold with K(m) approximately 116 microM K(+). The ATPase activity was moderately sensitive to ouabain and to SCH 28080 with apparent K(i) values in K(+)-free medium of approximately 64 microM and approximately 93 microM, respectively. Potassium exhibited strong antagonism toward both inhibitors. Assays of the ouabain-sensitive ATPase activity revealed inhibitory effects of Na(+) with the apparent K(i) of approximately 24 mM in the absence of added K(+) and with K(i) within the range of 60-70 mM in the presence of > or = 1 mM K(+). Thus, the human nongastric H,K-ATPase represented by the recombinant Atp1al1-betaHK complex exhibits enzymatic properties of K(+)-dependent ATPase sensitive to ouabain, SCH 28080, and Na(+). It differs from Na,K-ATPase in cation dependence and differs from gastric H,K-ATPase and Na,K-ATPase in sensitivity to inhibitors.     

Deceleration of the E1P-E2P transition and ion transport by mutation of potentially salt bridge-forming residues Lys-791 and Glu-820 in gastric H+/K+-ATPase

A lysine residue within the highly conserved center of the fifth transmembrane segment in P_IIC-type ATPase α-subunits is uniquely found in H,K-ATPases instead of a serine in all Na,K-ATPase isoforms. Because previous studies suggested a prominent role of this residue in determining the electrogenicity of non-gastric H,K-ATPase and in pK_a modulation of the proton-translocating residues in the gastric H,K-ATPases as well, we investigated its functional significance for ion transport by expressing several Lys-791 variants of the gastric H,K-ATPase in Xenopus oocytes. Although the mutant proteins were all detected at the cell surface, none of the investigated mutants displayed any measurable K⁺-induced stationary currents. In Rb⁺ uptake measurements, replacement of Lys-791 by Arg, Ala, Ser, and Glu substantially impaired transport activity and reduced the sensitivity toward the E₂-specific inhibitor {"type":"entrez-protein","attrs":{"text":"SCH28080","term_id":"1053015931","term_text":"SCH28080"}}SCH28080. Furthermore, voltage clamp fluorometry using a reporter site in the TM5/TM6 loop for labeling with tetra-methylrhodamine-6-maleimide revealed markedly changed fluorescence signals. All four investigated mutants exhibited a strong shift toward the E₁P state, in agreement with their reduced {"type":"entrez-protein","attrs":{"text":"SCH28080","term_id":"1053015931","term_text":"SCH28080"}}SCH28080 sensitivity, and an about 5–10-fold decreased forward rate constant of the E₁P ↔ E₂P conformational transition, thus explaining the E₁P shift and the reduced Rb⁺ transport activity. When Glu-820 in TM6 adjacent to Lys-791 was replaced by non-charged or positively charged amino acids, severe effects on fluorescence signals and Rb⁺ transport were also observed, whereas substitution by aspartate was less disturbing. These results suggest that formation of an E₂P-stabilizing interhelical salt bridge is essential to prevent futile proton exchange cycles of H⁺ pumping P-type ATPases.     

Mechanisms of urinary K+ and H+ excretion: primary structure and functional expression of a novel H,K-ATPase

The kidney plays an essential role in regulating potassium and acid balance. A major site for these regulations is in the collecting tubule. In the present study, we report the primary sequence of a novel alpha subunit of the P-ATPase gene family, which we isolated from the urinary bladder epithelium of the toad Bufo marinus, the amphibian equivalent of the mammalian collecting tubule. The cDNA encodes a protein of 1,042 amino acids which shares approximately 67% identity with the alpha 1 subunit of the ouabain-inhibitable Na,K-ATPase and approximately 69% identity with the alpha subunit of the SCH28080- inhibitable gastric H,K-ATPase. When coexpressed in Xenopus oocytes with a beta subunit isolated from the same cDNA library, the ATPase is able to transport rubidium (a potassium surrogate) inward, and hydrogen outward, leading to alkalization of the intracellular compartment and acidification of the external medium. The novel ATPase has a unique pharmacological profile showing intermediate sensitivity to both ouabain and SCH28080. Our findings indicate that the bladder ATPase is a member of a new ion motive P-ATPase subfamily. The bladder ATPase is expressed in the urinary tract but not in the stomach or the colon. This H,K-ATPase may be one of the molecules involved in H+ and K+ homeostasis, mediating the transport of these ions across urinary epithelia and therefore regulating their urinary excretion.     

The non-gastric H,K-ATPase is oligomycin-sensitive and can function as an H+,NH4(+)-ATPase

We used the baculovirus/Sf9 expression system to gain new information on the mechanistic properties of the rat non-gastric H,K-ATPase, an enzyme that is implicated in potassium homeostasis. The alpha2-subunit of this enzyme (HKalpha2) required a beta-subunit for ATPase activity thereby showing a clear preference for NaKbeta1 over NaKbeta3 and gastric HKbeta. NH4(+), K+, and Na+ maximally increased the activity of HKalpha2-NaKbeta1 to 24.0, 14.2, and 5.0 micromol P(i) x mg(-1) protein x h(-1), respectively. The enzyme was inhibited by relatively high concentrations of ouabain and SCH 28080, whereas it was potently inhibited by oligomycin. From the phosphorylation level in the presence of oligomycin and the maximal NH4(+)-stimulated ATPase activity, a turnover number of 20,000 min(-1) was determined. All three cations decreased the steady-state phosphorylation level and enhanced the dephosphorylation rate, disfavoring the hypothesis that Na+ can replace H+ as the activating cation. The potency with which vanadate inhibited the cation-activated enzyme decreased in the order K+ > NH4(+) > Na+, indicating that K+ is a stronger E2 promoter than NH4(+), whereas in the presence of Na+ the enzyme is in the E1 form. For K+ and NH4(+), the E2 to E1 conformational equilibrium correlated with their efficacy in the ATPase reaction, indicating that here the transition from E2 to E1 is rate-limiting. Conversely, the low maximal ATPase activity with Na+ is explained by a poor stimulatory effect on the dephosphorylation rate. These data show that NH4(+) can replace K+ with similar affinity but higher efficacy as an extracellular activating cation in rat nongastric H,K-ATPase.     

A chimeric gastric H+,K+-ATPase inhibitable with both ouabain and SCH 28080

2-Methyl-8-(phenylmethoxy)imidazo(1,2-a)pyridine-3acetonitrile+ ++ (SCH 28080) is a K+ site inhibitor specific for gastric H+,K+-ATPase and seems to be a counterpart of ouabain for Na+,K+-ATPase from the viewpoint of reaction pattern (i.e. reversible binding, K+ antagonism, and binding on the extracellular side). In this study, we constructed several chimeric molecules between H+,K+-ATPase and Na+,K+-ATPase alpha-subunits by using rabbit H+,K+-ATPase as a parental molecule. We found that the entire extracellular loop 1 segment between the first and second transmembrane segments (M1 and M2) and the luminal half of the M1 transmembrane segment of H+, K+-ATPase alpha-subunit were exchangeable with those of Na+, K+-ATPase, respectively, preserving H+,K+-ATPase activity, and that these segments are not essential for SCH 28080 binding. We found that several amino acid residues, including Glu-822, Thr-825, and Pro-829 in the M6 segment of H+,K+-ATPase alpha-subunit are involved in determining the affinity for this inhibitor. Furthermore, we found that a chimeric H+,K+-ATPase acquired ouabain sensitivity and maintained SCH 28080 sensitivity when the loop 1 segment and Cys-815 in the loop 3 segment of the H+,K+-ATPase alpha-subunit were simultaneously replaced by the corresponding segment and amino acid residue (Thr) of Na+,K+-ATPase, respectively, indicating that the binding sites of ouabain and SCH 28080 are separate. In this H+, K+-ATPase chimera, 12 amino acid residues in M1, M4, and loop 1-4 that have been suggested to be involved in ouabain binding of Na+, K+-ATPase alpha-subunit are present; however, the low ouabain sensitivity indicates the possibility that the sensitivity may be increased by additional amino acid substitutions, which shift the overall structural integrity of this chimeric H+,K+-ATPase toward 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.     

The human non-gastric H,K-ATPase has a different cation specificity than the rat enzyme

The primary sequence of non-gastric H,K-ATPase differs much more between species than that of Na,K-ATPase or gastric H,K-ATPase. To investigate whether this causes species-dependent differences in enzymatic properties, we co-expressed the catalytic subunit of human non-gastric H,K-ATPase in Sf9 cells with the beta(1) subunit of rat Na,K-ATPase and compared its properties with those of the rat enzyme (Swarts et al., J. Biol. Chem. 280, 33115-33122, 2005). Maximal ATPase activity was obtained with NH(4)(+) as activating cation. The enzyme was also stimulated by Na(+), but in contrast to the rat enzyme, hardly by K(+). SCH 28080 inhibited the NH(4)(+)-stimulated activity of the human enzyme much more potently than that of the rat enzyme. The steady-state phosphorylation level of the human enzyme decreased with increasing pH, [K(+)], and [Na(+)] and nearly doubled in the presence of oligomycin. Oligomycin increased the sensitivity of the phosphorylated intermediate to ADP, demonstrating that it inhibited the conversion of E(1)P to E(2)P. All three cations stimulated the dephosphorylation rate dose-dependently. Our studies support a role of the human enzyme in H(+)/Na(+) and/or H(+)/NH(4)(+) transport but not in Na(+)/K(+) transport.     

SCH28080, a K+-competitive inhibitor of the gastric H,K-ATPase,binds near the M5-6 luminal loop,preventing K+ access to the ion binding domain

Inhibition of the gastric H,K-ATPase by the imidazo[1,2-alpha]pyridine, SCH28080, is strictly competitive with respect to K+ or its surrogate, NH4+. The inhibitory kinetics [V(max), K(m,app)(NH4+), K(i)(SCH28080), and competitive, mixed, or noncompetitive] of mutants can define the inhibitor binding domain and the route to the ion binding region within M4-6. While mutations Y799F, Y802F, I803L, S806N, V807I (M5), L811V (M5-6), Y928H (M8), and Q905N (M7-8) had no effect on inhibitor kinetics, mutations P798C, Y802L, P810A, P810G, C813A or -S, I814V or -F, F818C, T823V (M5, M5-6, and M6), E914Q, F917Y, G918E, T929L, and F932L (M7-8 and M8) reduced the affinity for SCH28080 up to 10-fold without affecting the nature of the kinetics. In contrast, the L809F substitution in the loop between M5 and M6 resulted in an approximately 100-fold decrease in inhibitor affinity, and substitutions L809V, I816L, Y925F, and M937V (M5-6, M6, and M8) reduced the inhibitor affinity by 10-fold, all resulting in noncompetitive kinetics. The mutants L811F, Y922I, and I940A also reduced the inhibitor affinity up to 10-fold but resulted in mixed inhibition. The mutations I819L, Q923V, and Y925A also gave mixed inhibition but without a change in inhibitor affinity. These data, and the 9-fold loss of SCH28080 affinity in the C813T mutant, suggest that the binding domain for SCH28080 contains the surface between L809 in the M5-6 loop and C813 at the luminal end of M6, approximately two helical turns down from the ion binding region, where it blocks the normal ion access pathway. On the basis of a model of the Ca-ATPase in the E2 conformation (PDB entry 1kju), the mutants that change the nature of the kinetics are arranged on one side of M8 and on the adjacent side of the M5-6 loop and M6 itself. This suggests that mutations in this region modify the enzyme structure so that K+ can access the ion binding domain even with SCH28080 bound.     

The binding of a K+ competitive ligand, 2-methyl,8-(phenylmethoxy)imidazo(1,2-a)pyridine 3-acetonitrile, to the gastric (H+ + K+)-ATPase

2-Methyl,8-(phenylmethoxy)imidazo(1,2-a)pyridine 3-acetonitrile (SCH 28080) is a freely reversible K+ site inhibitor of the gastric (H+ + K+)-ATPase. In the presence of 2 mMMgSO4, [14C]SCH 28080 bound saturably to gastric vesicle preparations containing the (H+ + K+)-ATPase and was displaced by lumenal K+. A binding stoichiometry of 2.2 +/- 0.1 mol of SCH 28080/mol of catalytic phosphorylation sites was observed. The affinity of SCH 28080 binding was increased approximately 10-fold (to 45 nM) in the presence of 2 mM ATP. High affinity binding also occurred with 2 microM ATP but not with up to 200 microM D-[beta, gamma-CH2]ATP, suggesting that high affinity binding was to a phosphorylated form of the enzyme. In the presence of ATP, the association rate constant was linearly related to the concentration of SCH 28080. However, the association and dissociation rates of SCH 28080 binding were slow, especially at low temperature (at 1.5 degrees C half-maximal binding of 50 nM SCH 28080 was calculated to occur after 232 s). Binding appeared to be predominantly entropy driven with a high activation energy (40 kJ/mol at 37 degrees C). In the absence of ATP, the association rate constant was not linearly related to the concentration of SCH 28080, suggesting that a conformational change in the enzyme was required before binding could occur.     

Amino acid substitutions of Na,K-ATPase conferring decreased sensitivity to cardenolides in insects compared to mammals

Mutagenesis analyses and a recent crystal structure of the mammalian Na,K-ATPase have identified amino acids which are responsible for high affinity binding of cardenolides (such as ouabain) which at higher doses block the enzyme in the phosphorylated state. Genetic analysis of the Na,K-ATPase of insects adapted to cardenolides in their food plants revealed that some species possess substitutions which confer strongly increased resistance to ouabain in the mammalian enzyme such as the substitution T797A or combined substitutions at positions 111 and 122. To test for the effect of these mutations against the background of insect Na,K-ATPase, we here expressed the ouabain sensitive Na,K-ATPase α-subunit of Drosophila melanogaster together with the β-subunit Nrv3 in baculovirus-infected Sf9 cells and introduced the substitutions N122H, T797A, Q111T-N122H, Q111V-N122H, all of which have been observed in cardenolide-adapted insects. While all constructs showed similar expression levels, ouabain affinity of mutated Na,K-ATPases was reduced compared to the wild-type fly enzyme. Ouabain sensitivity of the ATPase activity in inhibition assays was significantly decreased by all mutations, yet whereas the IC₅₀ for the single mutations of N122H (61.0 μM) or T797A (63.3 μM) was increased roughly 250-fold relative to the wild-type (0.24 μM), the double mutations of Q111V-N122H (IC₅₀ 550 μM) and Q111T-N122H (IC₅₀ 583 μM) proved to be still more effective yielding a 2.250-fold increased resistance to ouabain. The double mutations identified in cardenolide-adapted insects are more effective in reducing ouabain sensitivity of the enzyme than those found naturally in the rat Na,K-ATPase (Q111R-N122D) or in mutagenesis screens of the mammalian enzyme. Obviously, the intense selection pressure on cardenolide exposed insects has resulted in very efficient substitutions that decrease cardenolide sensitivity extremely.     

Conversion of the low affinity ouabain-binding site of non-gastric H,K-ATPase into a high affinity binding site by substitution of only five amino acids

P-type ATPases of the IIC subfamily exhibit large differences in sensitivity toward ouabain. This allows a strategy in which ouabain-insensitive members of this subfamily are used as template for mutational elucidation of the ouabain-binding site. With this strategy, we recently identified seven amino acids in Na,K-ATPase that conferred high affinity ouabain binding to gastric H,K-ATPase (Qiu, L. Y., Krieger, E., Schaftenaar, G., Swarts, H. G. P., Willems, P. H. G. M., De Pont, J. J. H. H. M., and Koenderink, J. B. (2005) J. Biol. Chem. 280, 32349-32355). Because important, but identical, amino acids were not recognized in that study, here we used the non-gastric H,K-ATPase, which is rather ouabain-insensitive, as template. The catalytic subunit of this enzyme, in which several amino acids from Na,K-ATPase were incorporated, was expressed with the Na,K-ATPase beta1 subunit in Xenopus laevis oocytes. A chimera containing 14 amino acids, located in M4, M5, and M6, which are unique to Na,K-ATPase, displayed high affinity ouabain binding. Four of these residues, all located in M5, appeared dispensable for high affinity binding. Individual mutation of the remaining 10 residues to their non-gastric H,K-ATPase counterparts yielded five amino acids (Glu312,Gly319, Pro778, Leu795, and Cys802) whose mutation resulted in a loss of ouabain binding. In a final gain-of-function experiment, we introduced these five amino acids in different combinations in non-gastric H,K-ATPase and demonstrated that all five were essential for high affinity ouabain binding. The non-gastric H,K-ATPase with these five mutations had a similar apparent affinity for ouabain as the wild type Na,K-ATPase and showed a 2000 times increased affinity for ouabain in the NH4+-stimulated ATPase activity in membranes of transfected Sf9 cells.     

K-induced alkalinization in all cell types of rabbit gastric glands: A novel K/H exchange mechanism

Digital image processing of the pH-sensitive dye BCECF was used to examine the effects of high [K] media on cytoplasmic pH (pHi) of individual cells within isolated rabbit gastric glands. When cells were acidified to pHi 6.5 from the resting pHi of 7.2-7.3 and then exposed to solution containing 77 mM K plus amiloride (to block Na/H exchange), recovery to pHi 7.0 was observed. This K-induced alkalinization occurred in all cell types of the gland, including cells within antral glands that were devoid of parietal cells (PC). This process was independent of extracellular Na and Cl and was unaffected by: 5 mM Ba or 200 microM bumetanide, or acute treatment with either 500 microM ouabain or 100 microM cimetidine, histamine or carbachol. SCH28080, which inhibits the PC H/K-ATPase when used in the low microM range of concentrations, blocked the K effect on pHi at 100 microM but was ineffective at 1 microM. A similar pHi recovery was also stimulated by Li, Cs (both 72 mM), and Tl (10 mM), in the order Li greater than K greater than Cs greater than Tl (all in the presence of amiloride), and these alkalinizations were also blocked by 100 microM SCH28080. Parallel experiments were performed to test the effect of these ions on 14[C]-aminopyrine accumulation, an index of acid secretion by the H/K-ATPase at the lumenal membrane of the PC. There was no correlation between the rates of cation-induced pHi recovery from an acid load and H secretion as measured by the accumulation of aminopyrine. We conclude that the K- (and Cs- and Li-) dependent pHi recovery is mediated by a novel cation/H exchange mechanism that is distinct from the PC H/K-ATPase.     

Lysine 480 is not an essential residue for ATP binding or hydrolysis by Na,K-ATPase.

Lysine 480 has been suggested to be essential for ATP binding and hydrolysis by Na,K-ATPase because it is labeled by reagents that are thought to react with the ATPase from within the ATP binding site. In order to test this hypothesis, Lys-480 was changed to Ala, Arg, or Glu by site-directed mutagenesis, and the resultant Na,K-ATPase molecules were expressed in yeast cells. The ATPase activity of each of the mutants was similar to the activity of the wild type enzyme indicating that Lys-480 is not essential for ATP hydrolysis. The binding of [3H]ouabain in both ATP-dependent and inorganic phosphate-dependent reactions was used to determine the apparent affinity of each mutant for ATP or Pi. The K0.5(ATP) for ouabain binding to phosphoenzyme formed from ATP was 1-3 microM for Lys-480, Arg-480, and Ala-480, whereas for Glu-480 the K0.5(ATP) was 18 microM. The K0.5(Pi) for ouabain binding to phosphoenzyme formed from inorganic phosphate was 16-28 microM for Lys-480, Arg-480, and Ala-480, but was 74 microM for Glu-480. The Kd for ouabain binding was similar for both the wild type and mutant Na,K-ATPase molecules (3-6 nM). These data indicate that the substitution of an acidic amino acid for lysine at position 480 appears to reduce the affinity of the Na,K-ATPase for both ATP and phosphate. It is concluded that Lys-480 is not essential for ATP binding or hydrolysis or for phosphate binding by Na,K-ATPase but is likely to be located within the ATP binding site of the Na,K-ATPase.     

Cation transport in vesicles from secreting rabbit stomach

K+ gradient-dependent rubidium flux in vesicles obtained from stimulated rabbit stomach distinguishes two cation pathways. Selective inhibition by vanadate and the (1,2-alpha)-imidazopyridine, SCH 28080 identifies one pathway as H,K-ATPase-mediated passive cation exchange. A second pathway, additive to the first, is inhibited by the protonophore, tetrachlorosalicylanilide and is identified as a K+ conductance pathway present in these vesicles. The conductance was limited to vesicle populations obtained from the stimulated rabbit gastric mucosa and was distributed into both a light microsomal fraction and a heavier membrane fraction. 86Rb+ transport through the cation conductance exhibited a trans-stimulated cation selectivity sequence of K+ greater than Rb+ = Cs+ much greater than Li+. Potential sensitive flux was inhibited by the cyanine dye 3,3'-dipropyl-2,2'-thiodicarbo cyanine iodide, Ba2+, quinine, and the guanidinium compound 1,8-bis-guanidinium-n-octane. The presence of the conductance was correlated with K+-dependent H+ transport which did not require prolonged equilibration in K+ medium for activation. A role for the stimulus-dependent K+ conductance in gastric acid secretion could be its provision of a pathway for net K+ movement to the luminal site of the H,K-ATPase.