Site-directed mutagenesis of cation coordinating residues in the gastric H,K-ATPase.

Site-mutations were introduced into putative cation binding site 1 of the H,K-ATPase at glu-797, thr-825, and glu-938. The side chain oxygen of each was not essential but the mutations produced different activation and inhibition kinetics. Site mutations thr-825 (ala, leu) and glu-938 (ala, gln) modestly decreased the apparent affinity to K+, while glu-797 (gln) was equivalent to wild type. As expected of competitive inhibition, mutations of thr-825 and glu-938 that decreased the apparent affinity for K+ also increased the apparent affinity for SCH28080. This is consistent with the participation of thr-825 and glu-938 in a cation binding domain. The sidechain geometry, but not the sidechain charge of glu-797, is essential to ATPase function as the site mutant glu-797 (gly) inactivated the H,K-ATPase, while glu-797 (gln) was active but the apparent affinity to SCH 28080 was decreased by four-fold. Lys-793, a unique residue of the H,K-ATPase, was essential for ATPase function. Since this residue is adjacent to site 1, the result suggests that charge pairing between lys-793 and residues at or near this site may be essential to ATPase function.

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.

Transmembrane carboxyl residues are essential for cation-dependent function in the gastric H,K-ATPase.

The K+-dependent ATPase activity of the H,K-ATPase was irreversibly inhibited by the carboxyl activating reagent, dicyclohexylcarbodiimide (DCCD). The inhibition was first order and displayed a concentration dependence with the K0.5 (DCCD) = 0.65 +/- 0.04 mM. KCl protected 70% of the ATPase activity from DCCD-dependent inhibition in a concentration-dependent manner with a K0.5 (K+) = 0.58 +/- 0.1 mM KCl. DCCD modification selectively inhibited the K+-dependent rather than ATP-dependent partial reactions including eosin fluorescence responses and ligand-stabilized initial tryptic cleavage patterns of the membrane-associated enzyme. DCCD modification also inhibited the binding of 86Rb+ and the fluorescent responses of the K+-competitive, fluorescent inhibitor 1-(2-methylphenyl)-4-methylamino-6-methyl-2, 3-dihydropyrrolo[3,2-c]quinoline. [14C]DCCD was incorporated into the H,K-ATPase in a time course identical to that describing the inactivation of the K+-dependent ATPase activity of the H,K-ATPase. A component of the [14C]DCCD incorporated into the H,K-ATPase was K+-sensitive where K+ reduced the [14C]DCCD incorporated into the enzyme by 1.6 nmol of [14C]DCCD/mg of protein. Membrane-associated tryptic peptides resolved from the [14C]DCCD-modified H,K-ATPase exhibited various K+ sensitivities with peptides at 23, 9.6, 8.2, 7.1, and 6.1 kDa containing 10-78%, 23-52%, 24-36%, 2%, and 3-4% K+-sensitivity, respectively. The N-terminal sequence of the K+-sensitive, approximately 23- and 9.6-kDa peptides was LVNE857, a C-terminal fragment of the ATPase alpha-subunit. The mass of the smaller peptide limited the residue assignment to the transmembrane M7/M8 domains and an intervening extracytoplasmic loop. An N-terminal sequence, SD840IM, was obtained from a 3.3-kDa, [14C]DCCD-labeled peptide resolved from a V8 digest of the partially purified alpha-subunit. This mass was sufficient to include LVNE but would exclude M8 and the intervening loop between M7 and M8. Glu857 is a unique residue present in each of the proteolytic preparations of the H,K-ATPase modified by [14C]DCCD. These data provide functional evidence of the selective inactivation of the K+-dependent partial reactions of the H,K-ATPase and show that Glu857 located at the M7 boundary in the C terminus of the pump molecule is a significant site of DCCD modification. These data are interpreted to indicate that this carboxyl residue is important for cation binding function.

Rubidium occlusion within tryptic peptides of the H,K-ATPase.

86Rb+ binding to the H,K-ATPase was measured in the Mg(2+)-vanadate-inhibited enzyme at 4 degrees C. The concentration dependence of 86Rb+ binding in detergent-free preparations exhibited two components, one saturable with a K0.5 (Rb+) of 0.76 +/- 0.3 mM and a binding capacity of 2626 +/- 690 pmol of Rb+/mg of protein and the second nonsaturable, but linearly dependent, upon the 86Rb+ concentration. The concentration dependence of 86Rb+ binding was unaffected by digitonin treatment with a K0.5 (Rb+) of 0.63 +/- 0.09 mM and a binding capacity of 2824 +/- 152 pmol of Rb+/mg of protein, but the amplitude of the nonsaturable component was eliminated. The level of 86Rb+ binding was optimized by vanadate and decreased by ADP and ATP, suggesting that cation binding is stabilized in the E2-like conformation and antagonized in the E1 conformation. The Rb(+)-dependent stabilization of the E2 enzyme conformation was confirmed from the fluorescent quench response of the fluorescein isothiocyanate (FITC)-labeled enzyme, where 86Rb+ bound to the FITC-labeled enzyme with a K0.5 = 0.85 +/- 0.3 mM and a saturable binding capacity of 2121 pmol of 86Rb+/mg of protein and quenched the FITC fluorescence with a K0.5(Rb+) of 3.6 +/- 0.3 mM. The K(+)-competitive inhibitor, 1-(2-methylphenyl)-4-methylamino-6-methyl-2,3-dihydropyrrolo[3,2-c ]quinoline (MDPQ), also quenched FITC fluorescence with a K0.5(MDPQ) of 24.5 +/- 0.6 microM and competitively inhibited 86Rb+ binding with a K*0.5 = 35.8 microM (MDPQ). The MDPQ-induced quench of FITC fluorescence at Lys517 within the cytoplasmic M4/M5 nucleotide domain and displacement of 86Rb+ from a functionally defined extracytoplasmic binding domain indicate that structural determinants of the E2 conformational state exist within both cytoplasmic and extracytoplasmic domains of the H,K-ATPase and thus provide evidence of concerted conformational changes between the nucleotide and cation binding domains within the FITC-labeled H,K-ATPase. Membrane-bound fragments of the H,K-ATPase were prepared by tryptic hydrolysis in KCl medium. Tryptic digestion rapidly inactivated the phosphoenzyme site with a time course where k = 0.25 +/- 0.04 min-1 but both 86Rb+ binding and MDPQ fluorescence responses were retained. The concentration dependence of 86Rb+ binding and Rb(+)-dependent MDPQ fluorescence responses in the trypsin-digested membranes were described by a single class of binding sites where K0.5 = 1.2 +/- 0.3 and 0.73 +/- 0.09 mM, respectively. The stability of the Rb+ and MDPQ sites suggest their locations are near or within the membrane and are inaccessible to trypsin attack.(ABSTRACT TRUNCATED AT 400 WORDS)

Apical membrane of the gastric parietal cell: water, proton, and nonelectrolyte permeabilities.

Gastric parietal cell apical membranes must protect the cell from the extremely low pH and wide variations in osmolality of the gastric juice. To characterize the permeability properties of gastric apical membranes, we have measured passive permeabilities to water, protons, NH3, and small nonelectrolytes of membrane vesicles derived from parietal cells of fasted animals and fed animals. Both preparations are known to be highly enriched in H+/K(+)-ATPase, the enzyme responsible for acidifying the gastric contents. The preparations behaved as single populations, and their permeability properties were similar in all respects, permitting pooling of the results. This similarity suggests that insertion of tubulovesicles into the apical membrane does not change the behavior of the lipid bilayer. Osmotic water permeability (Pf) averaged (mean +/- SD) (2.8 +/- 0.3) x 10(-4) cm/s, a value 10-fold lower than that obtained in lecithin large unilamellar vesicles (LUV) and similar to that obtained in other water-tight epithelia. Similarly, ammonia permeability (PNH3) was low [(4.4 +/- 2.3) x 10(-3) cm/s] and 10 times below that of lecithin LUV. By contrast, proton permeability (PH+) was surprisingly high (0.030 +/- 0.011 cm/s) and similar to that of lecithin LUV. These results suggest that the pathway for proton permeation differs from that of water and NH3. Nonelectrolyte permeabilities were strikingly similar to those obtained in another water-tight epithelium, the toad urinary bladder. Moreover, these permeabilities followed Overton's rule in that permeability varied in accordance with the oil-water partition coefficient. We conclude that the gastric apical membrane, like that of several renal epithelia, is relatively water-tight and exhibits low permeabilities to small nonelectrolytes. These properties are likely to be essential to the ability of this membrane to perform its barrier function.

A K+ competitive, conformational probe of the H,K-ATPase.

The H,K-ATPase was noncovalently labelled with a fluorescent quinoline derivative, 1-(2-methylphenyl)-4-methylamino-6-methyl-2,3-dihydropyrrolo [3,2-c]quinoline, (MDPQ). MDPQ competitively inhibited the K+ stimulated ATP hydrolysis with a Ki of 0.22 microM but did not inhibit the MgATP-dependent phosphoenzyme to an extent greater than 10% of control. Inhibitor binding to the H,K-ATPase enhanced MDPQ fluorescence. This fluorescence was quenched by lumenal K+ with a K0.5 of 1.8 mM. MDPQ binding to the H,K-ATPase shifted the fluorescence Ex/Em maxima from 342/478 nm to 342/453 nm. Phosphorylation of the H,K-ATPase by MgATP further enhanced fluorescence with a difference spectra [MgATP-(MgATP+KCl)] emission peak at 446 nm. Trypsin dependent proteolysis of the H,K-ATPase stabilized within the E2K conformation eliminated the phosphoenzyme response, but enhanced the K+ specific dephosphoenzyme response. These observations show that MDPQ is a fluorescent, competitive inhibitor of the H,K-ATPase that interacts with a lumenal cation binding site. Under specific conditions, both the cation and MDPQ binding sites remain intact within trypsin produced cleavage peptides of the H,K-ATPase.

Conformational transitions of the H,K-ATPase studied with sodium ions as surrogates for protons.

Following a recent demonstration that H,K-ATPase can active transport Na+ at a low rate (Polvani, C., Sachs, G., and Blostein, R. (1989) J. Biol. Chem. 264, 17854-17859), we have looked for and found effects of Na+ ions on the conformational state of gastric H,K-ATPase labeled with fluorescein isothiocyanate. Na+ ions reverse the K(+)-induced quench of the fluorescein fluorescence and somewhat enhance fluorescence in the absence of K+ ions. Equilibrium titrations of the cation effects show that Na+ and K+ ions are strictly competitive with apparent dissociation constants of KNa+ = 62 mM (n = 2) and KK+ = 6.6 mM (n = 2). The observations demonstrate that Na+ ions bind to and stabilize the high fluorescence E1 form of the protein while K+ ions stabilize the low fluorescence E2 form. Elevation of pH from 6.4 to 8.0 increased the apparent affinity of the Na+ ions from approximately 62 to 10.2 mM, consistent with competition between protons and Na+. The action of Na+ to stabilize the E1 form was used to measure the rate of the E2K----E1Na transition with a stopped-flow fluorimeter. The rate at pH 6.4 and 20 degrees C is 18.1 s-1. In addition the rate of the reverse conformational transition E1K----E2K has been measured at several K+ concentrations. From the hyperbolic dependence on K+ concentration a maximal rate of 211 +/- 32 s-1 and intrinsic K+ dissociation constant on E1 of 64.6 +/- 3.3 mM have been estimated. The kinetic and equilibrium data are self-consistent and thus support the proposed action of Na+ and K+ ions. Compared with Na,K-ATPase, the H,K-ATPase exhibits a lower affinity for Na+ on E1 and a much faster rate of the E2K----E1Na transition, but a similar affinity for K+ ions on E1 and rate of the transition E1K----E2K. The significance of the similarities and differences in cation specificity and rates of conformational changes of Na,K- and H,K-ATPases is discussed.

Glutaraldehyde crosslinking analysis of the C12E8 solubilized H,K-ATPase.

A soluble porcine H,K-ATPase preparation was obtained with the nonionic detergent, C12E8. ATP hydrolysis by the soluble H,K-ATPase was stimulated with respect to the native preparation at pH 6.1, while the K(+)-phosphatase activity was comparable to the native enzyme. The soluble enzyme demonstrated characteristic ligand-dependent effects on ATP hydrolysis, including ATP activation of K(+)-stimulated hydrolysis with a K0.5 of 28 +/- 4 microM ATP, and inhibition with an IC50 of 2.1 mM ATP. The activation and inhibition of ATP hydrolysis by K+ was also observed with a K0.5 for activation of 2.8 +/- 0.4 mM KCl at 2.0 mM ATP (pH 6.1) and inhibition with an IC50 of 135 mM KCl at 0.05 mM ATP. 2-Methyl-8-(phenylmethoxy)imidazo[1,2a]pyridine-3-acetonitrile (SCH 28080), a specific inhibitor of the native H,K-ATPase, competitively inhibited the K(+)-stimulated activity with a Ki of 0.035 microM. The soluble enzyme was stable with a t0.5 for ATPase activity of 6 h between 4 and 11 degrees C. The demonstration of these related ligand responses in the catalytic reactions of the soluble preparation indicates that it is an appropriate medium for investigation of the subunit associations of the functional H,K-ATPase. Subunit associations of the active soluble enzyme were assessed following treatment with the crosslinking reagent, glutaraldehyde. The distribution of crosslinked particles was independent of the soluble protein concentration in the crosslinking buffer within the protein range 0.3 to 2.0 mg/ml or the detergent to protein ratio varied from 1 to 15 (w/w). The crosslinked pattern was unaffected by the presence or absence of K during crosslinking or nucleotide concentration. These observations suggest that crosslinking occurs in associated subunits that do not undergo rapid associations dependent upon enzyme turnover. Phosphorylation of the soluble enzyme with 0.1 mM MgATP produced a phosphoprotein at 94 kDa. A phosphoprotein obtained after glutaraldehyde treatment exhibited identical electrophoretic mobility to the crosslinked particle identified by silver stain. Glutaraldehyde treatment of soluble protein fractions resolved on a linear 10-35% glycerol gradient revealed several smaller peptides partially resolved from the crosslinked pump particle, but no active fraction enriched in the monomeric H,K-ATPase. This data indicates that the functional porcine gastric H,K-ATPase is organized as a structural dimer.

Radiation inactivation analysis of oligomeric structure of the H,K-ATPase.

The oligomeric size of the H,K-ATPase was determined in frozen gastric microsomal vesicles irradiated with high energy electrons. Target sizes of various catalytic activities associated with H,K-ATPase function fell into two distinct groups. The lower group of target sizes described the radiation-induced loss of steady-state phosphoenzyme and structural monomer: the MgATP-dependent formation of a beta-aspartyl phosphate exhibited a size range of 133-147 kDa; the size range for the structural measurement (i.e. loss of H,K-ATPase monomer on sodium dodecyl sulfate-polyacrylamide gels) was 92-143 kDa. In contrast, a larger group of target sizes described the loss of full cycle catalytic activities (i.e. K+-dependent stimulation of p-nitrophenyl phosphate and ATP hydrolysis). The K+-phosphatase and K+-stimulated ATPase exhibited target sizes fo 200 +/- 13 and 232 +/- 23 kDa, respectively. The lower target size group represents the first evidence that a monomer of the catalytic subunit maintains partial enzyme function. The larger group of target sizes describing K+-phosphatase and ATPase activities suggest that subunit interactions contribute to full cycle catalytic activity. Subunit interactions appear to be involved in all ion transport activities. Passive Rb+ exchange and active H+ transport in reconstituted proteoliposomes exhibited target sizes of 233n = 2 and 388 +/- 48 kDa, respectively. H+ transport appears to require a subunit arrangement more complex than that associated with catalytic activity or passive ion transport.