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J Biol Chem, Vol. 274, Issue 11, 6848-6854, March 12, 1999

A Chimeric Gastric H⁺,K⁺-ATPase Inhibitable with Both Ouabain and SCH 28080

From the Molecular Genetics Research Center and the ^§ Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama 930-0194, Japan

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 -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 -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 -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 -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 -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.

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