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

2-Methyl-8-(phenylmethoxy) imidazo( 1,2-a) pyridine-3-acetonitrile (SCH 280 80) is a K+ site inhibitor specific for gastric H+,K+-ATPase and seems to b e a counterpart of ouabain for Na+,K+-ATPase from the viewpoint of reaction pattern (i.e. reversible binding, K+ antagonism, and binding on the extrac ellular side). In this study, we constructed several chimeric molecules bet ween H+,K+-ATPase and Na+,K+-ATPase alpha-subunits by using rabbit H+,K+-AT Pase 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-sub unit were exchangeable with those of Na+,K+-ATPase, respectively, preservin g 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 fo und that a chimeric H+,K+-ATPase acquired ouabain sensitivity and maintaine d SCH 28080 sensitivity when the loop I segment and Cys-815 in the loop 3 s egment of the H+,K+-ATPase cu-subunit were simultaneously replaced by the c orresponding segment and amino acid residue (Thr) of Na+,K+-ATPase, respect ively, indicating that the binding sites of ouabain and SCH 28080 are separ ate. In this H+,K+-ATPase chimera, 12 amino acid residues in M1, M4, and lo op 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 ind icates the possibility that the sensitivity may be increased by additional amino acid substitutions, which shift the overall structural integrity of t his chimeric H+,K+-ATPase toward that of Na+,K+-ATPase.