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.     

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.     

Spectrophotometric method for the determination of renal ouabain-sensitive H+,K+-ATPase activity

The aim of this work was to develop a method for renal H+,K+-ATPase measurement based on the previously used Na+,K+-ATPase assay (Beltowski et al.: J Physiol Pharmacol.; 1998, 49: 625-37). ATPase activity was assessed by measuring the amount of inorganic phosphate liberated from ATP by isolated microsomal fraction. Both ouabain-sensitive and ouabain-resistant K+-stimulated and Na+-independent ATPase activity was detected in the renal cortex and medulla. These activities were blocked by 0.2 mM imidazolpyridine derivative, Sch 28080. The method for ouabain-sensitive H+,K+-ATPase assay is characterized by good reproducibility, linearity and recovery. In contrast, the assay for ouabain-resistant H+,K+-ATPase was unsatisfactory, probably due to low activity of this enzyme. Ouabain-sensitive H+,K+-ATPase was stimulated by K+ with Km of 0.26 +/- 0.04 mM and 0.69 +/- 0.11 mM in cortex and medulla, respectively, and was inhibited by ouabain (Ki of 2.9 +/- 0.3 microM in the renal cortex and 1.9 +/- 0.4 microM in the renal medulla) and by Sch 28080 (Ki of 1.8 +/- 0.5 microM and 2.5 +/- 0.9 microM in cortex and medulla, respectively). We found that ouabain-sensitive H+,K+-ATPase accounted for about 12% of total ouabain-sensitive activity in the Na+,K+-ATPase assay. Therefore, we suggest to use Sch 28080 during Na+,K+-ATPase measurement to block H+,K+-ATPase and improve the assay specificity. Leptin administered intraperitoneally (1 mg/kg) decreased renal medullary Na+,K+-ATPase activity by 32.1% at 1 h after injection but had no effect on H+,K+-ATPase activity suggesting that the two renal ouabain-sensitive ATPases are separately regulated.     

Properties of light and heavy vesicles simultaneously prepared from hog gastric mucosae

We obtained two kinds of vesicle preparations which were of different density from the same gastric mucosae of hogs stimulated with food before slaughter. Both kinds contained H+,K+-ATPase. The light vesicle preparation differed from the heavy vesicle preparation as follows: the KCl permeability across the membrane of heavy vesicles was larger than that of light vesicles, the actin (46-kDa peptide on SDS-polyacrylamide gel) content of heavy vesicles was much higher than that of light vesicles, and the H+,K+-ATPase activity of heavy vesicles was less sensitive to a monoclonal antibody raised against light vesicles (HK2032) than that of light vesicles. Furthermore, there was a drastic difference in reactivity to SCH 28080, which is an H+,K+-ATPase-specific inhibitor and reacts competitively with the K+-high affinity site. SCH 28080 is more potent in light vesicles than in heavy vesicles. These results suggest that the conformation of H+,K+-ATPase changed during the translocation from tubulovesicles to the apical plasma membrane. On the other hand, H+,K+-ATPase activities in both vesicles had similar pH and [K+] dependences.     

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.     

The colonic H+,K+-ATPase functions as a Na+-dependent K+(NH4+)-ATPase in apical membranes from rat distal colon.

Recent studies have suggested that the colonic H+,K+-ATPase (HKalpha2) can secrete either Na+ or H+ in exchange for K+. If correct, this view would indicate that the transporter could function as either a Na+ or a H+ pump. To investigate this possibility a series of experiments was performed using apical membranes from rat colon which were enriched in colonic H+,K+-ATPase protein. An antibody specific for HKalpha2 was employed to determine whether HKalpha2 functions under physiological conditions as a Na+-dependent or Na+-independent K+-ATPase in this same membrane fraction. K+-ATPase activity was measured as [gamma-32P]ATP hydrolysis. The Na+-dependent K+-ATPase accounted for approximately 80% of overall K+-ATPase activity and was characterized by insensitivity to Sch-28080 but partial sensitivity to ouabain. The Na+-independent K+-ATPase activity was insensitive to both Sch-28080 and ouabain. Both types of K+-ATPase activity substituted NH4+ for K+ in a similar manner. Furthermore, our results demonstrate that when incubated with native distal colon membranes, the blocking antibody inhibited dramatically Na+-dependent K+-ATPase activity. Therefore, these data demonstrate that HKalpha2 can function in native distal colon apical membranes as a Na+-dependent K+-ATPase. Elucidation of the role of the pump as a transporter of Na+ versus H+ or NH4+ versus K+ in vivo will require additional studies.     

Identification of an extracytoplasmic region of H+,K(+)-ATPase labeled by a K(+)-competitive photoaffinity inhibitor.

The photoaffinity reagent 8-[(4-azidophenyl)-methoxy]-1-tritiomethyl-2, 3-dimethylimidazo-[1,2-alpha]pyridinium iodide ([3H]mDAZIP) has been synthesized and used to photoinactivate and label purified hog gastric H+,K(+)-ATPase. The specific (K(+)-sensitive) components of both photoinactivation and labeling showed dependences on inhibitor concentration consistent with covalent modification at an extracytoplasmic site of reversible K(+)-competitive binding in the dark. The maximum amount of specific labeling (1.2 nmol/mg) was similar to the number of phosphorylation sites measured (1.0 +/- 0.14 nmol/mg). Specific labeling was distributed 76% on the alpha chain, 18% on the beta chain, and 6% on undefined peptides. Various digestions with trypsin, protease V8, and thermolysin were employed to fragment the labeled enzyme. Gasphase sequencing of the radioactive peptides identified the major site of specific labeling to be within a region where only two stretches of amino acids (Leu105 to Ile126 and Leu139 to Phe155, designated H1 and H2, respectively) are predicted to span the membrane. This in turn suggested that the labeling site was located within or close to the proposed loop between them (Gln127 to Asn138). A computer-driven energy minimization protocol yielded a loop structure to which SCH 28080 (the parent structure of [3H]mDAZIP) could be docked. Conversely, modeling of the corresponding region of Na+,K(+)-ATPase (a homologous enzyme with much lower affinity for SCH 28080) yielded no apparent binding site. Similarities in the inhibition of H+,K(+)-ATPase by SCH 28080 and of Na+,K(+)-ATPase by ouabain lead to the hypothesis that, in each case, inhibitor binding to E2-P is associated with an increase in the hydrophobicity of the environment of the loop between H1 and H2.     

Phosphorylation of H+/K(+)-ATPase by inorganic phosphate. The role of K+ and SCH 28080.

The effects of K+ on the phosphorylation of H+/K(+)-ATPase with inorganic phosphate were studied using H+/K(+)-ATPase purified from porcine gastric mucosa. The phosphoenzyme formed by phosphorylation with Pi was identical with the phosphoenzyme formed with ATP. The maximal phosphorylation level obtained with Pi was equal to that obtained with ATP. The Pi phosphorylation reaction of H+/K(+)-ATPase was, like that of Na+/K(+)-ATPase, a relatively slow reaction. The rates of phosphorylation and dephosphorylation were both increased by low concentrations of K+, which resulted in hardly any effect on the phosphorylation level. A decrease of the steady-state phosphorylation level was caused by higher concentrations of K+ in a noncompetitive manner, whereas no further increase in the dephosphorylation rate was observed. The decreasing effect was caused by a slow binding of K+ to the enzyme. All above-mentioned K+ effects were abolished by the specific H+/K(+)-ATPase inhibitor SCH 28080 (2-methyl-8-[phenyl-methoxy]imidazo-[1-2-a]pyrine-3-acetonitrile). Additionally, SCH 28080 caused a 2-fold increase in the affinity of H+/K(+)-ATPase for Pi. A model for the reaction cycle of H+/K(+)-ATPase fitting the data is postulated.     

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.     

Inhibition of gastric H+,K+-ATPase and acid secretion by SCH 28080, a substituted pyridyl(1,2a)imidazole

A hydrophobic amine, SCH 28080, 2-methyl-8-(phenylmethoxy)imidazo(1,2a)pyridine-3-acetonitrile, previously shown to inhibit gastric acid secretion in vivo and in vitro, was also shown to inhibit basal and stimulated aminopyrine accumulation in isolated gastric glands when histamine, high K+ concentrations, or dibutyryl cAMP were used as secretagogues. Stimulated, but not basal, oxygen consumption was also inhibited. Neutralization of the acid space of the parietal cell by high concentrations of the weak base, imidazole, reduced the potency of the drug, suggesting that SCH 28080 was active when protonated. Studies on the isolated H+,K+-ATPase showed that the compound inhibited the enzyme competitively with K+, whether ATP or p-nitrophenyl phosphate were used as substrates. In contrast, the inhibition was mixed with respect to p-nitrophenyl phosphate and uncompetitive with respect to ATP. The drug reduced the steady state level of the phosphoenzyme but not the observed rate constant for phosphoenzyme formation in the absence of K+ nor the quantity of phosphoenzyme reacting with K+. The drug quenched the fluorescence of fluorescein isothiocyanate-modified enzyme and also inhibited the ATP-independent K+ exchange reaction of the H+,K+-ATPase. Its action on gastric acid secretion can be explained by inhibition of the H+,K+-ATPase by reversible complexation of the enzyme. This class of compound, therefore, acts as a reversible inhibitor of gastric acid secretion.     

Functional association between K+-Cl- cotransporter-4 and H+,K+-ATPase in the apical canalicular membrane of gastric parietal cells

We studied whether K+-Cl(-) cotransporters (KCCs) are involved in gastric HCl secretion. We found that KCC4 is expressed in the gastric parietal cells more abundantly at the luminal region of the gland than at the basal region. KCC4 was found in the stimulation-associated vesicles (SAV) derived from the apical canalicular membrane but not in the intracellular tubulovesicles, whereas H+,K+-ATPase was expressed in both of them. In contrast, KCC1, KCC2, and KCC3 were not found in either SAV or tubulovesicles. KCC4 coimmunoprecipitated with H+,K+-ATPase in the lysate of SAV. Interestingly the MgATP-dependent uptake of (36)Cl(-) into the SAV was suppressed by either the H+,K+-ATPase inhibitor (SCH28080) or the KCC inhibitor ((R)-(+)-[(2-n-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy]acetic acid). The KCC inhibitor suppressed the H+ uptake into SAV and the H+,K+-ATPase activity of SAV, but the inhibitor had no effects on these activities in the freeze-dried leaky SAV. These results indicate that the K+-Cl(-) cotransport by KCC4 is tightly coupled with H+/K+ antiport by H+,K+-ATPase, resulting in HCl accumulation in SAV. In the tetracycline-regulated expression system of KCC4 in the HEK293 cells stably expressing gastric H+,K+-ATPase, KCC4 was coimmunoprecipitated with H+,K+-ATPase. The rate of recovery of intracellular pH in the KCC4-expressing cells after acid loading through an ammonium pulse was significantly faster than that in the KCC4-non-expressing cells. Our results suggest that KCC4 and H+,K+-ATPase are the main machineries for basal HCl secretion in the apical canalicular membrane of the resting parietal cell. They also may contribute in part to massive acid secretion in the stimulated state.     

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.     

Leishmania plasma membrane Mg2+-ATPase is a H+/K+-antiporter involved in glucose symport. Studies with sealed ghosts and vesicles of opposite polarity

Experiments from other laboratories conducted with Leishmania donovani promastigote cells had earlier indicated that the plasma membrane Mg2+-ATPase of the parasite is an extrusion pump for H+. Taking advantage of the pellicular microtubular structure of the plasma membrane of the organism, we report procedures for obtaining sealed ghost and sealed everted vesicle of defined polarity. Rapid influx of H+ into everted vesicles was found to be dependent on the simultaneous presence of ATP (1 mm) and Mg2+ (1 mm). Excellent correspondence between rate of H+ entry and the enzyme activity clearly demonstrated the Mg2+-ATPase to be a true H+ pump. H+ entry into everted vesicle was strongly inhibited by SCH28080 (IC50 = approximately 40 microm) and by omeprazole (IC50 = approximately 50 microm), both of which are characteristic inhibitors of mammalian gastric H+,K+-ATPase. H+ influx was completely insensitive to ouabain (250 microm), the typical inhibitor of Na+,K+-ATPase. Mg2+-ATPase activity could be partially stimulated with K+ (20 mm) that was inhibitable (>85%) with SCH28080 (50 microm). ATP-dependent rapid efflux of 86Rb+ from preloaded vesicles was completely inhibited by preincubation with omeprazole (150 microm) and by 5,5'-dithiobis-(2-nitrobenzoic acid) (1 mm), an inhibitor of the enzyme. Assuming Rb+ to be a true surrogate for K+, an ATP-dependent, electroneutral stoichiometric exchange of H+ and K+(1:1) was established. Rapid and 10-fold active accumulation of [U-(14)C]2-deoxyglucose in sealed ghosts could be observed when an artificial pH gradient (interior alkaline) was imposed. Rapid efflux of [U-(14)C]d-glucose from preloaded everted vesicles could also be initiated by activating the enzyme, with ATP. Taken together, the plasma membrane Mg2+-ATPase has been identified as an electroneutral H+/K+ antiporter with some properties reminiscent of the gastric H+,K+-ATPase. This enzyme is possibly involved in active accumulation of glucose via a H+-glucose symport system and in K+ accumulation.     

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.     

A hybrid between Na+,K+-ATPase and H+,K+-ATPase is sensitive to palytoxin, ouabain, and SCH 28080

Na(+),K(+)-ATPase is inhibited by cardiac glycosides such as ouabain, and palytoxin, which do not inhibit gastric H(+),K(+)-ATPase. Gastric H(+),K(+)-ATPase is inhibited by SCH28080, which has no effect on Na(+),K(+)-ATPase. The goal of the current study was to identify amino acid sequences of the gastric proton-potassium pump that are involved in recognition of the pump-specific inhibitor SCH 28080. A chimeric polypeptide consisting of the rat sodium pump alpha3 subunit with the peptide Gln(905)-Val(930) of the gastric proton pump alpha subunit substituted in place of the original Asn(886)-Ala(911) sequence was expressed together with the gastric beta subunit in the yeast Saccharomyces cerevisiae. Yeast cells that express this subunit combination are sensitive to palytoxin, which interacts specifically with the sodium pump, and lose intracellular K(+) ions. The palytoxin-induced K(+) efflux is inhibited by the sodium pump-specific inhibitor ouabain and also by the gastric proton pump-specific inhibitor SCH 28080. The IC(50) for SCH 28080 inhibition of palytoxin-induced K(+) efflux is 14.3 +/- 2.4 microm, which is similar to the K(i) for SCH 28080 inhibition of ATP hydrolysis by the gastric H(+),K(+)-ATPase. In contrast, palytoxin-induced K(+) efflux from cells expressing either the native alpha3 and beta1 subunits of the sodium pump or the alpha3 subunit of the sodium pump together with the beta subunit of the gastric proton pump is inhibited by ouabain but not by SCH 28080. The acquisition of SCH 28080 sensitivity by the chimera indicates that the Gln(905)-Val(930) peptide of the gastric proton pump is likely to be involved in the interactions of the gastric proton-potassium pump with SCH 28080.     

Constitutive activation of gastric H+,K+-ATPase by a single mutation.

In the reaction cycle of P-type ATPases, an acid-stable phosphorylated intermediate is formed which is present in an intracellularly located domain of the membrane-bound enzymes. In some of these ATPases, such as Na+,K+-ATPase and gastric H+, K+-ATPase, extracellular K+ ions stimulate the rate of dephosphorylation of this phosphorylated intermediate and so stimulate the ATPase activity. The mechanism by which extracellular K+ ions stimulate the dephosphorylation process is unresolved. Here we show that three mutants of gastric H+,K+-ATPase lacking a negative charge on residue 820, located in transmembrane segment six of the alpha-subunit, have a high SCH 28080-sensitive, but K+-insensitive ATPase activity. This high activity is caused by an increased 'spontaneous' rate of dephosphorylation of the phosphorylated intermediate. A mutant with an aspartic acid instead of a glutamic acid residue in position 820 showed hardly any ATPase activity in the absence of K+, but K+ ions stimulated ATPase activity and the dephosphorylation process. These findings indicate that the negative charge normally present on residue 820 inhibits the dephosphorylation process. K+ ions do not stimulate dephosphorylation of the phosphorylated intermediate directly, but act by neutralizing the inhibitory effect of a negative charge in the membrane.     

Characterization of gill (Na+ + K+)-ATPase in the sea bass (Dicentrarchus labrax L.)

Bass gill microsomal preparations contain both a Na+, K+ and Mg2+-dependent ATPase, which is completely inhibited by 10(-3)M ouabain and 10(-2)M Ca2+, and also a ouabain insensitive ATP-ase activity in the presence of both Mg2+ and Na+. Under the optimal conditions of pH 6.5, 100 mM Na+, 20 mM K+, 5 mM ATP and 5 mM Mg2+, (Na+ + K+)-ATPase activity at 30 degrees C is 15.6 mumole Pi hr/mg protein. Bass gill (Na+ + K+)-ATPase is similar to other (Na+ + K+)-ATPases with respect to the sensitivity to ionic strength, Ca2+ and ouabain and to both Na+/K+ and Mg2+/ATP optimal ratios, while pH optimum is lower than poikilotherm data. The enzyme requires Na+, whereas K+ can be replaced efficiently by NH+4 and poorly by Li+. Both Km and Vm values decrease in the series NH+4 greater than K+ greater than Li+. The break of Arrhenius plot at 17.7 degrees C is close to the adaptation temperature. Activation energies are scarcely different from each other and both lower than those generally reported. The Km for Na+ poorly decreases as the assay temperature lowers. The comparison with literature data aims at distinguishing between distinctive and common features of bass gill (Na+ + K+)-ATPase.     

Studies on K+ permeability of rat gastric microsomes

A population of gastric membrane vesicles of high K+ permeability and of lower density than endoplasmic tubulovesicles containing (H+-K+)-ATPase was detected in gastric mucosal microsomes from the rat fasted overnight. The K+-transport activity as measured with 86RbCl uptake had a Km for Rb+ of 0.58 +/- 0.11 mM and a Vmax of 13.7 +/- 1.9 nmol/min X mg of protein. The 86Rb uptake was reduced by 40% upon substituting Cl- with SO2-4 and inhibited noncompetitively by ATP and vanadate with a Ki of 3 and 30 microM, respectively; vanadate also inhibited rat gastric (H+-K+)-ATPase but with a Ki of 0.03 microM. Carbachol or histamine stimulation decreased the population of the K+-permeable light membrane vesicles, at the same time increased K+-transport activity in the heavy, presumably apical membranes of gastric parietal cells, and enabled the heavy microsomes to accumulate H+ ions in the presence of ATP and KCl without valinomycin. The secretagogue-induced shift of K+ permeability was blocked by cimetidine, a H2-receptor antagonist. Four characteristics of the K+ permeability as measured with 86RbCl were common in the resting light and the carbachol-stimulated heavy microsomes; (a) Km for +Rb, (b) anion sensitivity (Cl- greater than SO2-4), (c) potency of various divalent cations (Hg2+, Cu2+, Cd2+, and Zn2+) to inhibit Rb+ uptake, and (d) inhibitory effect of ATP, although the nucleotide sensitivity was latent in the stimulated heavy microsomes. The Vmax for 86RbCl uptake was about 10 times greater in the resting light than the stimulated heavy microsomes. These observations led us to propose that secretagogue stimulation induces the insertion of not only the tubulovesicles containing (H+-K+)-ATPase, but also the light membrane vesicles containing KCl transporter into the heavy apical membranes of gastric parietal cells.     

Photoaffinity labeling of the lumenal K+ site of the gastric (H+ + K+)-ATPase

A photoaffinity label for the lumenal K+ site of the gastric (H+ + K+)-ATPase has been identified. Seven azido derivatives based upon the reversible K+ site inhibitor SCH 28080 were studied, one of which, m-ATIP (8-(3-azidophenylmethoxy)-1,2,3-trimethylimidazo[1,2-a] pyridinium iodide), was subsequently synthesized in radiolabeled form. In the absence of UV irradiation, m-ATIP inhibited K+ -stimulated ATPase activity in lyophilized gastric vesicles competitively with respect to K+, with a Ki value of 2.4 microM at pH 7.0. Irradiation of lyophilized gastric vesicles at pH 7.0 with [14C]m-ATIP in the presence of 0.2 mM ATP resulted in a time-dependent inactivation of ATPase activity that was associated with an incorporation of radioactivity into a 100-kDa polypeptide representing the catalytic subunit of the (H+ + K+)-ATPase. Both inactivation and incorporation were blocked in the presence of 10 mM KCl but not with 10 mM NaCl, consistent with interaction at the K+ site. The level of incorporation required to produce complete inhibition of ATPase activity was 1.9 +/- 0.2 times the number of catalytic phosphorylation sites in the same preparation. Tryptic digestion of gastric vesicle membranes, labeled with [14C]m-ATIP, failed to release the radioactivity from the membranes suggesting that the site of interaction was close to or within the membrane-spanning sections of this ion pump.     

A model of reversible inhibitors in the gastric H+/K+-ATPase binding site determined by rotational echo double resonance NMR

Several close analogues of the noncovalent H(+)/K(+)-ATPase inhibitor SCH28080 (2-methyl-3-cyanomethyl-8-(phenylmethoxy)imidazo[1,2-a]pyridine) have been screened for activity and examined in the pharmacological site of action by solid-state NMR spectroscopy. TMPIP, the 1,2,3-trimethyl analogue of SCH28080, and variants of TMPIP containing fluorine in the phenylmethoxy ring exhibited IC(50) values for porcine H(+)/K(+)-ATPase inhibition falling in the sub-10 microm range. Deuterium NMR spectra of a (2)H-labeled inhibitor titrated into H(+)/K(+)-ATPase membranes revealed that 80-100% of inhibitor was bound to the protein, and K(+)-competition (2)H NMR experiments confirmed that the inhibitor lay within the active site. The active binding conformation of the pentafluorophenylmethoxy analogue of TMPIP was determined from (13)C-(19)F dipolar coupling measurements using the cross-polarization magic angle spinning NMR method, REDOR. It was found that the inhibitor adopts an energetically favorable extended conformation falling between fully planar and partially bowed extremes. These findings allowed a model to be proposed for the binding of this inhibitor to H(+)/K(+)-ATPase based on the results of independent site-directed mutagenesis studies. In the model, the partially bowed inhibitor interacts with Phe(126) close to the N-terminal membrane spanning helix M1 and residues in the extracellular loop bridging membrane helices M5 and M6 and is flanked by residues in M4.