Gastric proton pump with two occluded K+ engineered with sodium pump-mimetic mutations.

The gastric H+,K+-ATPase mediates electroneutral exchange of 1H+/1K+ per ATP hydrolysed across the membrane. Previous structural analysis of the K+-occluded E2-P transition state of H+,K+-ATPase showed a single bound K+ at cation-binding site II, in marked contrast to the two K+ ions occluded at sites I and II of the closely-related Na+,K+-ATPase which mediates electrogenic 3Na+/2K+ translocation across the membrane. The molecular basis of the different K+ stoichiometry between these K+-counter-transporting pumps is elusive. We show a series of crystal structures and a cryo-EM structure of H+,K+-ATPase mutants with changes in the vicinity of site I, based on the structure of the sodium pump. Our step-wise and tailored construction of the mutants finally gave a two-K+ bound H+,K+-ATPase, achieved by five mutations, including amino acids directly coordinating K+ (Lys791Ser, Glu820Asp), indirectly contributing to cation-binding site formation (Tyr340Asn, Glu936Val), and allosterically stabilizing K+-occluded conformation (Tyr799Trp). This quintuple mutant in the K+-occluded E2-P state unambiguously shows two separate densities at the cation-binding site in its 2.6 Å resolution cryo-EM structure. These results offer new insights into how two closely-related cation pumps specify the number of K+ accommodated at their cation-binding site.

Gastric-type H+,K+-ATPase in mouse vestibular end organs.

CONCLUSION: Gastric type H+,K+-ATPase in the vestibular end organs may be of importance for K+ circulation and may also be related to pH regulation in vestibular end organs and endolymphatic sac. OBJECTIVE: To analyze the expression of gastric-type H+,K+-ATPase in normal mouse vestibular end organs. METHODS: 8 weeks old CBA/J mice were used in this study. The presence of gastric-type H+,K+-ATPase α and ß in the vestibular end organs, viz. utricle, saccule, ampulla, vestibular ganglion, and endolymphatic sac, was investigated using immunohistochemistry. RESULTS: In the vestibular end organs, H+,K+-ATPase α and ß were almost identical. H+,K+-ATPase was expressed in sensory cells, the basolateral surface of dark cells, fibrocytes, in vestibular ganglion cells, and in the upper region of the endolymphatic sac epithelial cells.

Isolation of H(+),K(+)-ATPase-enriched Membrane Fraction from Pig Stomachs.

Gastric H(+),K(+)-ATPase is an ATP-driven proton pump responsible for the acid secretion. Here, we describe the procedure for the isolation of H(+),K(+)-ATPase-enriched membrane vesicle fractions by Ficoll/sucrose density gradient centrifugation. Further purification by SDS treatment of membrane fractions is also introduced. These procedures allow us to obtain purified protein preparations in a quantity of several tens of milligrams, with the specific activity of ~480 µmol/mg/h. High purity and stability of H(+),K(+)-ATPase in the membrane preparation enable us to evaluate its detailed biochemical properties, and also to obtain 2D crystals for structural analysis.

Expression of Na,K-ATPase and H,K-ATPase Isoforms with the Baculovirus Expression System.

P-type ATPases can be expressed in several cell systems. The baculovirus expressions system uses an insect virus to enter and express proteins in Sf9 insect cells. This expression system is a lytic system in which the cells will die a few days after viral infection. Subsequently, the expressed proteins can be isolated. Insect cells are a perfect system to study P-type ATPases as they have little or no endogenous Na,K-ATPase activity and other ATPase activities can be inhibited easily. Here we describe in detail the expression and isolation of Na,K-ATPase and H,K-ATPase isoforms with the baculovirus expression system.

Active detergent-solubilized H+,K+-ATPase is a monomer.

The H(+),K(+)-ATPase pumps protons or hydronium ions and is responsible for the acidification of the gastric fluid. It is made up of an α-catalytic and a ß-glycosylated subunit. The relation between cation translocation and the organization of the protein in the membrane are not well understood. We describe here how pure and functionally active pig gastric H(+),K(+)-ATPase with an apparent Stokes radius of 6.3 nm can be obtained after solubilization with the non-ionic detergent C(12)E(8), followed by exchange of C(12)E(8) with Tween 20 on a Superose 6 column. Mass spectroscopy indicates that the ß-subunit bears an excess mass of 9 kDa attributable to glycosylation. From chemical analysis, there are 0.25 g of phospholipids and around 0.024 g of cholesterol bound per g of protein. Analytical ultracentrifugation shows one main complex, sedimenting at s(20,)(w) = 7.2 ± 0.1 S, together with minor amounts of irreversibly aggregated material. From these data, a buoyant molecular mass is calculated, corresponding to an H(+),K(+)-ATPase α,ß-protomer of 147.3 kDa. Complementary sedimentation velocity with deuterated water gives a picture of an α,ß-protomer with 0.9-1.4 g/g of bound detergent and lipids and a reasonable frictional ratio of 1.5, corresponding to a Stokes radius of 7.1 nm. An α(2),ß(2) dimer is rejected by the data. Light scattering coupled to gel filtration confirms the monomeric state of solubilized H(+),K(+)-ATPase. Thus, α,ß H(+),K(+)-ATPase is active at least in detergent and may plausibly function as a monomer, as has been established for other P-type ATPases, Ca(2+)-ATPase and Na(+),K(+)-ATPase.

Purification and characterization of the ouabain-sensitive H+/K+-ATPase from guinea-pig distal colon.

Distal colon absorbs K+ through a Na+-independent, ouabain-sensitive H+/K+-exchange, associated to an apical ouabain-sensitive H+/K+-ATPase. Expression of HKalpha2, gene associated with this ATPase, induces K+-transport mechanisms, whose ouabain susceptibility is inconsistent. Both ouabain-sensitive and ouabain-insensitive K+-ATPase activities have been described in colonocytes. However, native H+/K+-ATPases have not been identified as unique biochemical entities. Herein, a procedure to purify ouabain-sensitive H+/K+-ATPase from guinea-pig distal colon is described. H+/K+-ATPase is Mg2+-dependent and activated by K+, Cs+ and NH4+ but not by Na+ or Li+, independently of K+-accompanying anion. H+/K+-ATPase was inhibited by ouabain and vanadate but insensitive to SCH-28080 and bafilomycin-A. Enzyme was phosphorylated from [32P]-gamma-ATP, forming an acyl-phosphate bond, in an Mg2+-dependent, vanadate-sensitive process. K+ inhibited phosphorylation, effect blocked by ouabain. H+/K+-ATPase is an alpha/beta-heterodimer, whose subunits, identified by Tandem-mass spectrometry, seems to correspond to HKalpha2 and Na+/K+-ATPase beta1-subunit, respectively. Thus, colonic ouabain-sensitive H+/K+-ATPase is a distinctive P-type ATPase.

Identification of the beta-subunit for nongastric H-K-ATPase in rat anterior prostate.

The structural organization of nongastric H-K-ATPase, unlike that of closely related Na-K-ATPase and gastric H-K-ATPase, is not well characterized. Recently, we demonstrated that nongastric H-K-ATPase alpha-subunit (alpha(ng)) is expressed in apical membranes of rodent prostate. Its highest level, as well as relative abundance, with respect to alpha(1)-isoform of Na-K-ATPase, was observed in anterior lobe. Here, we aimed to determine the subunit composition of nongastric H-K-ATPase through the detailed analysis of the expression of all known X-K-ATPase beta-subunits in rat anterior prostate (AP). RT-PCR detects transcripts of beta-subunits of Na-K-ATPase only. Measurement of absolute protein content of these three beta-subunit isoforms, with the use of quantitative Western blotting of AP membrane proteins, indicates that the abundance order is beta(1) > beta(3) >> beta(2). Immunohistochemical experiments demonstrate that beta(1) is present predominantly in apical membranes, coinciding with alpha(ng), whereas beta(3) is localized in the basolateral compartment, coinciding with alpha(1). This is the first direct demonstration of the alpha(ng)-beta(1) colocalization in situ indicating that, in rat AP, alpha(ng) associates only with beta(1). The existence of alpha(ng-)beta(1) complex has been confirmed by immunoprecipitation experiments. These results indicate that beta(1)-isoform functions as the authentic subunit of Na-K-ATPase and nongastric H-K-ATPase. Putatively, the intracellular polarization of X-K-ATPase isoforms depends on interaction with other proteins.

Glycosylation is essential for biosynthesis of functional gastric H+,K+-ATPase in insect cells.

The role of N-linked glycosylation in the functional properties of gastric H+,K+-ATPase has been examined with tunicamycin and I-deoxymannojirimycin, inhibitors in glycoprotein biosynthesis and glycoprotein processing respectively. Tunicamycin completely abolished both K+-stimulated and 3-(cyanomethyl)-2-methyl-8-(phenylmethoxy)-imidazo[1,2a]pyridine (SCH 28080)-sensitive ATPase activity and SCH 28080-sensitive phosphorylation capacity. The expression level of both H+,K+-ATPase subunits remained unaffected. 1-Deoxymannojirimycin clearly affected the structure of the N-linked oligosaccharide moieties without affecting specific phosphorylation capacity. Purification of the functional recombinant enzyme from non-functional H+,K+-ATPase subunits coincided with purification of glycosylated beta-subunits and not of non-glycosylated beta-subunits. Transport of the H+,K+-ATPase beta-subunit to the plasma membrane but not its ability to assemble with the alpha-subunit dependent on N-glycosylation events. We conclude that the acquisition, but not the exact structure, of N-linked oligosaccharide moieties, is essential for biosynthesis of functional gastric H+,K+-ATPase in insect cells.

Evidence for a selective and electroneutral K+/H(+)-exchange in Saccharomyces cerevisiae using plasma membrane vesicles.

The existence of a K+/H+ transport system in plasma membrane vesicles from Saccharomyces cerevisiae is demonstrated using fluorimetric monitoring of proton fluxes across vesicles (ACMA fluorescence quenching). Plasma membrane vesicles used for this study were obtained by a purification/reconstitution protocol based on differential and discontinuous sucrose gradient centrifugations followed by an octylglucoside dilution/gel filtration procedure. This method produces a high percentage of tightly-sealed inside-out plasma membrane vesicles. In these vesicles, the K+/H+ transport system, which is able to catalyse both K+ influx and efflux, is mainly driven by the K+ transmembrane gradient and can function even if the plasma membrane H(+)-ATPase is not active. Using the anionic oxonol VI and the cationic DISC2(5) probes, it was shown that a membrane potential is not created during K+ fluxes. Such a dye response argues for the presence of a K+/H+ exchange system in S. cerevisiae plasma membrane and established the non-electrogenic character of the transport. The maximal rate of exchange is obtained at pH 6.8. This reversible transport system presents a high selectivity for K+ among other monovalent cations and a higher affinity for the K+ influx into the vesicles (exit from cells). The possible role of this K+/H+ exchange system in regulation of internal potassium concentration in S. cerevisiae is discussed.

Characterization of Syrian hamster gastric mucosal H+,K+-ATPase.

Employing a simple one-step sucrose gradient fractionation method, gastric mucosal membrane of Syrian hamster was prepared and demonstrated to be specifically enriched in H+,K+-ATPase activity. The preparation is practically devoid of other ATP hydrolyzing activity and contains high K+-stimulated ATPase activity of at least 4-5 fold compared to basal ATPase activity. The H+,K+-ATPase showed hydroxylamine-sensitive phosphorylation and K+-dependent dephosphorylation of the phosphoenzyme, characteristic inhibition by vanadate, omeprazole and SCH 28080, and nigericin-reversible K+-dependent H+-transport--properties characteristic of gastric proton pump. One notable difference with H+,K+-ATPase of other species has been the observation of valinomycin-independent H+ transport in such membrane vesicles. It is proposed that such H+,K+-ATPase-rich hamster gastric mucosal membrane preparation might provide a unique model to study physiological aspects of H+,K+-ATPase function in relation to HCl secretion.

The conformations of a functional spin-labeled derivative of gastric H/K-ATPase investigated by EPR spectroscopy.

A spin-labeled derivative of porcine gastric H/K-ATPase with high ATP hydrolyzing activity (77 mumol of Pi/(mg.h)) has been prepared. Over 65% of initial ATPase activity (115 mumol of Pi/(mg.h)) was preserved after complete reaction of the enzyme with the lysine reactive nitroxide spin-labeled TEMPO isothiocyanate (TITC). In contrast, rapid and complete loss of ATPase activity occurred after reaction of the enzyme with the lysine directed fluorescent probe FITC. Conventional EPR spectra of TITC labeled H/K-ATPase reflected mainly the slow rotational diffusion of the enzyme in the membrane. An upper limit enzyme intramembranous radius of 108 A was calculated on the basis of rotational correlation times estimated from saturation transfer (ST) EPR spectral lineshapes. Conventional EPR spectra exhibited two major components corresponding to at least two populations of strongly constrained spin-labels. Difference spectroscopy revealed that the proportion of these two components changed markedly with temperature. Moreover, the proportion of the components was sensitive to the presence of the activating ionic ligands Mg2+ and ATP, which induce enzyme conformational transitions, and to the reversible inhibitor SCH 28080, which binds to the K+ sensitive form of the enzyme. These findings show that EPR spectroscopy is able to report functionally coupled conformational changes of gastric H/K-ATPase and imply that the spin-labels are attached to lysines within functionally important regions of the enzyme.

Structure-activity relationships of unsaturated long chain fatty acids in relation to the inhibition of gastric H+,K(+)-ATPase.

Structure-activity analyses of the effects of unsaturated fatty acids on the inhibition of gastric H+,K(+)-ATPase were carried out. Saturated fatty acids are poor inhibitors of H+,K(+)-ATPase, compared with unsaturated ones. Among the cis-unsaturated fatty acids with one double bond, there was an optimal carbon chain length for the inhibitory of the enzyme, and 10-nonadecenoic acid (C19:1) was the most potent inhibitor. The inhibitory activity was not altered by a change in the position of the double bond. The cis-fatty acids were more potent than the corresponding trans-fatty acids. Esterification of the acid moiety resulted in a decrease of the inhibitory activity. However, conversion of the acid moiety into alcohol and amide did not decrease the activity. These findings suggest that two moieties of unsaturated fatty acids are important in the interaction with the gastric H+,K(+)-ATPase, the carbon chain, which may have some hydrophobic interactions with the enzyme, and the terminal functional group (acid moiety), which may interact with hydrophilic interactions such as through hydrogen bonds with the enzyme.

Assembly of Na,K-ATPase alpha-subunit isoforms with Na,K-ATPase beta-subunit isoforms and H,K-ATPase beta-subunit.

cDNA encoding an epitope tag was joined to cDNAs encoding the chicken Na,K-ATPase beta 1 and beta 2 and H,K-ATPase beta-subunits to allow recognition of these beta-subunits with the same monoclonal antibody during assembly assays. cDNAs encoding chicken Na,K-ATPase alpha 1, alpha 2, or alpha 3 and Na,K-ATPase beta 1 or beta 2 or H,K-ATPase beta-subunits were transiently coexpressed in mammalian cells. Subunit assembly was assayed by immune precipitation of alpha-isoforms with a monoclonal antibody to the epitope-tagged beta-subunits. Each of the chicken alpha-isoforms assembled with each of the Na,K-ATPase beta-subunits and the H,K-ATPase beta-subunit. Each of the epitope-tagged beta-subunits also assembled with a Na,K-ATPase/Ca-ATPase chimera that retained only 26 amino acids of the Na,K-ATPase alpha-subunit, demonstrating that all three beta-subunits recognize this same alpha-subunit assembly site.

Three-dimensional (type I) microcrystals of detergent-solubilized membrane-bound gastric (H+, K+)-ATPase enzyme from hog and rabbit stomachs.

Multilamellar 3-dimensional (Type I) microcrystals of detergent-solubilized crude microsomes or purified protein preparations of membrane-bound gastric (H+, K+)-ATPase from rabbit or hog stomachs develop in media consisting of 0.1 M KCl, 20 mM imidazole, 5 mM MgCl2, 3 mM NaN3, 5 mM DTT, 25 IU/ml Trasylol, 2 micrograms/ml DTBpC and 20-40% glycerol, using nonionic detergent of C12E8 or BRIJ 36 for solubilization. Crystals developed in a pH-range of 6.0-7.25, during 3-10 days of incubation, at 2 degrees C. For C12E8, the most effective detergent:protein ratio for crystallization varied between (1.8-2.0):1 for the microsomes and between (0.25-0.75):1 for the purified preparations. The results of biochemical and structural analysis of the (H+, K+)-ATPase crystals showed close resemblance to those of the sarcoplasmic reticulum Ca(2+)-ATPase from skeletal muscle and plasmamembrane (Na+, K+)-ATPase from kidney (J. Biol. Chem., 269, 10107-111, 1994). Based on these identities and the high (62%) overall sequential homology to the (Na+, K+)-ATPase, we conclude that the new crystals of the (H+, K+)-ATPase could also contain only the alpha-chain of the alpha beta heterodimers found in the native membrane. High-resolution electron microscopy of frozen-hydrated crystalline (H+, K+)-ATPase samples are in progress to give unit cell dimensions and molecular packing of the new crystals.

Stimulus-associated protein in gastric parietal cell detected using antimelittin antibody.

The bee venom polypeptide melittin binds to and inhibits the gastric hydrogen-potassium-adenosinetriphosphatase (H(+)-K(+)-ATPase). A search for parietal cell proteins with a melittin-like structure was carried out. A 67-kDa (doublet) protein, which reacted with a polyclonal antimelittin antibody, was found in purified rabbit parietal cells. The protein exhibited reversible stimulus-dependent redistribution from cytosol to (total) membranes. It was also found to be associated with H(+)-K(+)-ATPase-containing membranes when isolated from the gastric mucosae of rabbits treated with histamine, but not with cimetidine. The presence of the protein correlated with the ability of the membrane preparations to exhibit ionophore-independent HCl accumulation, a characteristic of gastric membranes from histamine-stimulated animals. The 67-kDa melittin-like protein may play a role in the functional changes in the gastric parietal cell that are involved in stimulation of HCl secretion.