IDENTIFICATION OF THE SITE OF INHIBITION BY OMEPRAZOLE OF A ALPHA-BETA FUSION PROTEIN OF THE H,K-ATPASE USING SITE-DIRECTED MUTAGENESIS

The alpha subunit of eukaryotic P-type ATPases has ten experimentally defined transmembrane or membrane inserted segments. The fifth and six th of these are short, not predicted by hydropathy analysis, do not in sert independently into microsomal membranes, and are readily removed after tryptic digestion and therefore may be membrane inserted sequenc es. Acid transport by the gastric H,K-ATPase is covalently inhibited b y several substituted pyridyl methylsulfinyl benzimidazoles, such as o meprazole. These act as probes of accessible extracytoplasmic thiols b ecause they are accumulated in the acid transporting gastric vesicles and then convert to thiol reactive, cationic tetracyclic sulfenamides. Inhibition is due mainly to disulfide formation with Cys(813) or Cys( 822) in M5/6 and perhaps with a contribution from Cys(892) in the loop between transmembrane segment (TM) 7 and TM8. Identification of the s pecific cysteine responsible for inhibition should be able to define t he turn between M5 and M6. The gastric H,K-ATPase alpha-beta heterodim er was expressed as a fusion protein in HEK 293 cells. Transient trans fection resulted in most of the protein being retained in the endoplas mic reticulum with only core glycosylation and minor activity of the A TPase evident. Stable transfection resulted in plasma membrane localiz ation of the protein and complex glycosylation, The transfected but no t the control cells displayed cation-stimulated, SCH 28080-inhibited A TPase activity and SCH 28080- and omeprazole-inhibited Rb-86 uptake. T he two cysteines in M5/6 and Cys(892) in the TM7/8 loop were mutated t o the amino acids found in the Na,K-ATPase in order to determine which of the three cysteine residues were important for benzimidazole inhib ition. Mutation of one, two, or all three cysteines did not alter enzy me activity, Rb-86 transport, or SCH 28080 inhibition. Only removal of Cys(822) blocked omeprazole inhibition of Rb-86 transport. These data suggest that Cys(822) is present in a region of the enzyme most easil y accessed by the cationic sulfenamide formed by omeprazole, presumabl y the turn between M5 and M6.