Amino acids Val115-Ile126 of rat gastric H(+)-K(+)-ATPase confer high affinity for Sch-28080 to Na(+)-K(+)-ATPase

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Abstract
Na(+)-K(+)-ATPase is inhibited by cardiac glycosides and is insensitive to Sch-28080, an inhibitor of gastric H(+)-K(+)-ATPase. Gastric H(+)-K(+)-ATPase is not inhibited by cardiac glycosides. Both ouabain and, Sch-28080 binding are inhibited by K+, and it has been suggested that the inhibitors bind to corresponding regions on the alpha-subunit of each ion pump. For identification of regions of each pump that interact with the specific inhibitors, chimeric alpha-subunits consisting of selected regions from Na(+)-K(+)-ATPase and gastric H(+)-K(+)-ATPase have been prepared. One chimera (gM1/2) has been constructed from cDNA of the sheep alpha1-subunit of Na(+)-K(+)-ATPase by replacement of the last 12 amino acids of the first predicted transmembrane region (Ile99-Ile110) with corresponding amino acids from rat gastric H(+)-K(+)-ATPase. gM1/2 was expressed in yeast cells together with either the rat Na(+)-K(+)-ATPase beta 1-subunit (NK beta 1) or rat gastric H(+)-K(+)-ATPase beta-subunit (HK beta). Western blots show that the expression level of the chimeric alpha-subunit was comparable to the Na(+)-K(+)-ATPase alpha 1. Ouabain binds with high affinity to gM1/2+NK beta 1 [ouabain binding affinity (Kd) = 9.5 nM] but not to gM1/2+HK beta. The Kd for ouabain binding to Na(+)-K(+)-ATPase was 7.8 nM. Na(+)-K(+)-ATPase activity of gM1/2+NK beta 1 was inhibited both by ouabain and Sch-28080. The 50% inhibition concentration for Sch-28080 was 20-60 nM. Sch-28080 at 10 microM did not inhibit Mg(2+)- and Pi-dependent ouabain binding to gM1/2+NK beta 1. Ouabain (0.75 mM) inhibited palytoxin-induced K+ efflux from yeast cells expressing either gM1/2+NK beta 1 or gM1/2+NK beta, and Sch-28080 increased the palytoxin-induced K+ efflux from yeast cells expressing gM1/2+NK beta 1 or gM1/2+HK beta. These results implicate a small number of amino acids in the first transmembrane part of gastric H(+)-K(+)-ATPase as partial determinants of the sensitivity to Sch-28080. The data also suggest that ouabain and Sch-28080 do not bind to the same site on the chimera.