Structural aspects of the gastric H,K ATPase.
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, cleaved with trypsin, and the membrane fragments compared before and after photolysis. Disappearance of the segment corresponding to TM3,4 and a relative loss of the segment corresponding to TM7,8 suggests close proximity of these two membrane pairs to the loop joining the fifth and sixth transmembrane segments, in particular TM3,4. Use of this type of covalent, photoactivatable site-specific reagent to determine loop proximity can be extended to other acid transporters.
Shin
J M
JM
Department of Physiology and Medicine, UCLA, USA.
Besancon
M
M
Bamberg
K
K
Sachs
G
G
D000595:Amino Acid Sequence; D000818:Animals; D000897:Anti-Ulcer Agents / Q000494:pharmacology; D001665:Binding Sites; D002462:Cell Membrane / Q000201:enzymology; D019281:Dimerization; D005753:Gastric Mucosa / Q000201:enzymology*; D017506:H(+)-K(+)-Exchanging ATPase / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010295:Parietal Cells, Gastric / Q000201:enzymology; D014176:Protein Biosynthesis; D017433:Protein Structure, Secondary; D051381:Rats
D000897:Anti-Ulcer Agents; D017506:H(+)-K(+)-Exchanging ATPase