A chimeric gastric H+,K+-ATPase inhibitable with both ouabain and SCH 28080

J Biol Chem. 1999 Mar 12;274(11):6848-54. doi: 10.1074/jbc.274.11.6848.

Abstract

2-Methyl-8-(phenylmethoxy)imidazo(1,2-a)pyridine-3acetonitrile+ ++ (SCH 28080) is a K+ site inhibitor specific for gastric H+,K+-ATPase and seems to be a counterpart of ouabain for Na+,K+-ATPase from the viewpoint of reaction pattern (i.e. reversible binding, K+ antagonism, and binding on the extracellular side). In this study, we constructed several chimeric molecules between H+,K+-ATPase and Na+,K+-ATPase alpha-subunits by using rabbit H+,K+-ATPase as a parental molecule. We found that the entire extracellular loop 1 segment between the first and second transmembrane segments (M1 and M2) and the luminal half of the M1 transmembrane segment of H+, K+-ATPase alpha-subunit were exchangeable with those of Na+, K+-ATPase, respectively, preserving H+,K+-ATPase activity, and that these segments are not essential for SCH 28080 binding. We found that several amino acid residues, including Glu-822, Thr-825, and Pro-829 in the M6 segment of H+,K+-ATPase alpha-subunit are involved in determining the affinity for this inhibitor. Furthermore, we found that a chimeric H+,K+-ATPase acquired ouabain sensitivity and maintained SCH 28080 sensitivity when the loop 1 segment and Cys-815 in the loop 3 segment of the H+,K+-ATPase alpha-subunit were simultaneously replaced by the corresponding segment and amino acid residue (Thr) of Na+,K+-ATPase, respectively, indicating that the binding sites of ouabain and SCH 28080 are separate. In this H+, K+-ATPase chimera, 12 amino acid residues in M1, M4, and loop 1-4 that have been suggested to be involved in ouabain binding of Na+, K+-ATPase alpha-subunit are present; however, the low ouabain sensitivity indicates the possibility that the sensitivity may be increased by additional amino acid substitutions, which shift the overall structural integrity of this chimeric H+,K+-ATPase toward that of Na+,K+-ATPase.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • Cell Line
  • DNA Primers
  • Enzyme Inhibitors / pharmacology*
  • H(+)-K(+)-Exchanging ATPase / genetics
  • Humans
  • Imidazoles / pharmacology*
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Ouabain / pharmacology*
  • Proton Pump Inhibitors*
  • Rabbits
  • Recombinant Fusion Proteins / antagonists & inhibitors*
  • Recombinant Fusion Proteins / genetics
  • Sequence Homology, Amino Acid
  • Stomach / enzymology*

Substances

  • DNA Primers
  • Enzyme Inhibitors
  • Imidazoles
  • Proton Pump Inhibitors
  • Recombinant Fusion Proteins
  • Sch 28080
  • Ouabain
  • H(+)-K(+)-Exchanging ATPase