The vesicular gastric H,K-ATPase catalyzes an electroneutral H for K exchange allowing acidification of the intravesicular space. There is a total of 28 cysteines present in the ␣ subunit of the gastric H,K-ATPase, of which 10 are found in the predicted transmembrane segments and their connecting loop, and 9 are present in the ␤ subunit, of which 6 are disulfide-linked. To determine which of these was accessible to extracytoplasmic attack, the enzyme was inhibited by four different sub- , under acid transporting conditions. All of these compounds are weak bases that accumulate in the acidic space generated by the pump and undergo an acid catalyzed rearrangement to a cationic sulfenamide, which forms disulfides with accessible cysteines. The relative rates of acid activation of these compounds corresponded to the relative rates of inhibition of ATPase activity and acid transport. Fragmentation of the enzyme by trypsin followed by SDS-polyacrylamide gel electrophoresis showed that omeprazole bound covalently to one of the two cysteines in the domains containing the fifth and sixth transmembrane segments and their extracytoplasmic loop and to cysteine 892 in the loop between the seventh and eighth transmembrane segments, but inhibition correlated with the reaction with cysteines in the fifth and sixth domain. Lansoprazole bound to the cysteines in these two domains as well as to cysteine 321 toward the extracytoplasmic end of the third transmembrane segments. Pantoprazole bound only to either cysteine 813 or 822 in the fifth and sixth transmembrane region. The inhibition of Rabeprazole correlated also with its binding to this part of the protein, but this compound continued to bind after full inhibition, eventually binding also to cysteines 321 and 892. No binding was found to any of the cysteines in the seventh to tenth transmembrane segments. Thermolysin digestion of the isolated omeprazole-labeled fifth and sixth transmembrane pair showed that cysteine 813 was the site of labeling. It is concluded that binding of these sided reagents to cysteine 813 in the loop between transmembrane (TM)5 and TM6 is sufficient for inhibition of ATPase activity and acid transport by the gastric acid pump. Of the 10 cysteines present in the membrane and extracytoplasmic domain, only three are exposed sufficiently to allow reactivity with these cationic thiol reagents. The binding to cysteine 813 defines the location of the extracytoplasmic loop between TM5 and TM6 and places the carboxylic acids 820 and 824 conserved between the gastric H,K-and the Na,K-ATPases in TM6, consistent with their assumed role in cation binding.

The gastric H+,K(+)-ATPase is an alpha beta heterodimer with close homology to the Na+,K(+)-ATPase. Digestion of intact cytoplasmic-side-out vesicles at a trypsin to protein ratio of 1/4 removed most of the cytoplasmic protein, leaving membrane-spanning pairs in high yield. These were visualized on gels and poly(vinylidene difluoride) (PVDF) membranes by sodium dodecyl sulfate solubilization of the membrane-embedded segments and labeling of the cysteine residues with fluorescein maleimide prior to electrophoresis. The membrane-spanning residues of the alpha subunit were found between positions 104 and 162 (M1/M2), 291 and 358(M3/M4), 776 and 835 (M5/M6), and 853 and 946 (M7/M8). Although this method did not detect membrane retention of the hydrophobic sequences subsequent to position 946, it provided biochemical evidence for at least eight membrane segments in the catalytic subunit. Intact vesicles containing this enzyme transport acid in the presence of KCl, valinomycin, and MgATP. Omeprazole accumulates in these acidified vesicles and converts to a cationic sulfenamide. This forms disulfides with accessible cysteines. The reaction with this extracytoplasmic thiol reagent inhibits ATPase activity. Full inhibition was obtained with a stoichiometry of 2.2 mol of omeprazole bound/mg of protein. Only the alpha subunit was labeled. The cysteines reacting with omeprazole were defined by proteolytic cleavage of 3H- or 14C-omeprazole-labeled enzyme followed by peptide sequencing of fragments separated on tricine gradient gels and transferred to PVDF membranes. Tryptic digestion at a 1/40 trypsin to protein ratio in the presence of ligands that stabilize the E2P form of the enzyme produced two large fragments, one of 68 kDa stretching from Glu47 to probably Arg666 that contained minor labeling and the other of 333 kDa beginning at Ala671 and extending to probably Arg946 that contained greater than 85% of the label. Digestion of labeled vesicles at 1/75 or 1/4 trypsin to protein ratios gave radioactive patterns consistent with labeling at Cys813 and/or Cys822 and at Cys892 and/or Cys927 and/or Cys938. V8 protease digestion of the solubilized alpha subunit produced a fragment extending from Ser838 to possible Asp900 that was omeprazole-labeled, showing that Cys892 was labeled and Cys927 and Cys938 were not. Hence, omeprazole labels the H+,K(+)-ATPase at cysteines within the M5/M6 and M7/M8 regions of the alpha subunit, accounting for its inhibitory action in vivo and in vitro.

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SUMMARY The synthesis and action of H2‐receptor antagonists changed the understanding of gastric acid secretion as well as changing medical therapy for peptic ulcer disease. It is now known that peripheral regulation of gastric acid secretion depends largely, but not entirely, on histamine release from the enterochromaffin‐like cell. There is, therefore, no final common pathway for stimulation of the parietal cell. In contrast, all stimuli converge to activate the acid pump, the H+, K+‐ATPase. Inhibition of this pump by clinically useful drugs was achieved by developing derivatives of timoprazole, pyridyl‐2‐methylsulftnyl benzimidazole. Two of these derivatives, omeprazole and lansoprazole, have shown superiority in acid control and therefore in therapy for peptic ulcer disease compared to the available H2‐receptor antagonists.

An H(+)-K(+)-adenosinetriphosphatase (H(+)-K(+)-ATPase) contributes to potassium reabsorption by the collecting ducts of the rat kidney. mRNAs for two isoforms of the H(+)-K(+)-ATPase, HK alpha 1 and HK alpha 2, have been found in the rat kidney. To evaluate whether the HK alpha 1 and HK alpha 2 proteins are present in the rat kidney, microsomes enriched in HK alpha 1 or HK alpha 2 were isolated using the MiniMac magnetic separation system with antibodies directed against either HK alpha 1 (HK 12.18) or HK alpha 2 (AS 31.7). Immunoblots of rat kidney microsomal protein isolated with HK 12.18 revealed a band approximately 94 kDa in size that comigrated with the G1 fraction of the stomach. Immunoblots of rat kidney microsomal protein isolated with AS 31.7 revealed a band slightly greater than 94 kDa that comigrated with a band obtained from rat colonic microsomal protein. To examine the effect of perturbations in potassium metabolism, the abundance of the HK alpha 1 and HK alpha 2 isoforms was compared in rats fed a normal or potassium-deficient diet. A low-potassium diet increased the abundance of HK alpha 2, whereas that of HK alpha 1 was not altered. These data suggest that HK alpha 2 might be the isoform responsible for potassium conservation by the kidney.

The gastric H,K ATPase is an alpha beta heterodimeric member of the eukaryotic alkali-cation P-type ion-motive ATPase family. The alpha subunit is composed of 1033 amino acids and the beta subunit of 291 amino acids with 6 or 7 potential N-linked glycosylation sites. Much effort has been expended to define the membrane domain of P-type ATPases. A membrane domain of the large subunit consisting of 10 membrane-spanning sequences is suggested by a combination of methods such as (1) tryptic digestion, separation, and sequencing of membrane peptides, (2) labeling with extracytoplasmic reagents, and (3) in vitro translation of hydrophobic segments. The beta subunit has a single transmembrane segment with strong hydrophobic interactions with the alpha subunit. Blue native gel electrophoresis shows that the enzyme is an (alpha-beta)2 dimer. Cross-linking with Cu-phenanthroline provides evidence that association is between the alpha subunits, and the potential SH groups that are Cu sensitive are at cysteine 565 and cysteine 615, in the region of the large cytoplasmic loop between the fourth and fifth transmembrane segments. No cross-linking is observed in the membrane domain. ATP prevents cross-linking because of a conformational change at the surface of the protein induced by ATP or by direct binding of the nucleotide at the site of cross-linking. The WGA binding properties of the beta subunit allow investigation of the region of interaction with the alpha subunit. Thus, digestion of the enzyme by trypsin followed by SDS solubilization and selective elution from a WGA column resulted in coelution of the membrane fragment containing TM7 and TM8. This result demonstrates major hydrophobic interaction between the seventh and eighth transmembrane segments and the beta subunit. An antibody generated against rat parietal cells also recognized shared epitopes in the same region of both the alpha and beta subunits. Biochemical investigation of the arrangement of the transmembrane segments has been hindered by the lack of effective cross-linking reagents probably because of the compact arrangement of this domain, preventing even Cu access. A series of antiulcer drugs has been developed that have a unique chemistry related to their inhibition of the gastric H,K ATPase. They are 2-(substituted pyridyl methylsulfinyl) benzimidazoles, weak bases with a pKa of 4.0. After accumulation in the acidic space generated by the H,K ATPase either in vivo or in vitro, they undergo acid-catalyzed conversion to a tetracyclic sulfenamide which reacts with luminally accessible SH residues to form stable disulfide derivatives. In the particular case of pantoprazole, 2-(3,4-dimethoxy-2-pyridyl-methylsulfinyl)-5-difluoromethoxy benzimidazole, reaction is confined largely to cysteine 813, placed between the fifth and sixth transmembrane segments. The 5 azido analog of pantoprazole provided acid transport-dependent inhibition of the isolated transporting ATPase by this photoactivatable covalent SH reagent. The inhibited enzyme was then photolyzed, cleav...

Both receptor antagonists and acid pump inhibitors are clinically useful suppressants of acid secretion. The latter class of drugs, the substituted benzimidazoles, inhibit acid secretion more effectively and, therefore, provide superior symptom relief and healing in all acid‐ related diseases. The H2‐receptor antagonists competitively block the action of histamine on the H2‐receptors of parietal cells. This histamine is released from enterochromaffin‐like cells (ECL cells) due to gastrin, acetylcholine or epinephrine stimulation. In addition, parietal cells have M3‐receptors which can function independently of H2‐ receptors. Hence, there is no single common pathway for parietal cell stimulation. Stimulation of acid secretion by parietal cells requires activation of the acid pump, the gastric H+,K(+)‐ATPase. The target site for the benzimidazoles is the activated gastric H+,K(+)‐ATPase, and, in particular, the cysteines of the pump that are exposed to the acid space of the secretory canaliculus of the parietal cells. Pantoprazole in its protonated form selectively reacts with cysteines present in both the fifth and sixth membrane segments of the ATPase, explaining its mechanism of inhibiting proton transport by this enzyme.