AMINO-ACIDS VAL(115)-ILE(126) OF RAT GASTRIC H-K+-ATPASE CONFER HIGH-AFFINITY FOR SCH-28080 TO NA+-K+-ATPASE()

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 bi nding are inhibited by K+, and it has been suggested that the inhibito rs bind to corresponding regions on the alpha-subunit of each ion pump . For identification of regions of each pump that interact with the sp ecific inhibitors, chimeric alpha-subunits consisting of selected regi ons from Na+-K+-ATPase and gastric H+-K+-ATPase have been prepared. On e chimera (gM1/2) has been constructed from cDNA of the sheep alpha(1) -subunit of Na+-K+-ATPase by replacement of the last 12 amino acids of the first predicted transmembrane region (Ile(99)-Ile(110)) with corr esponding amino acids from rat gastric H+-K+-ATPase. gM1/2 was express ed in yeast cells together with either the rat Na+-K+-ATPase beta(1)-s ubunit (NK beta(1)) or rat gastric H+-K+-ATPase beta-subunit (HK beta) . Western blots show that the expression level of the chimeric alpha-s ubunit was comparable to the Na+-K+-ATPase alpha(1). Ouabain binds wit h high affinity to gM1/2+NK beta 1 [ouabain binding affinity (K-d) = 9 .5 nM] but not to gM1/2+HK beta. The K-d for ouabain binding to Na+-K-ATPase was 7.8 nM. Na+-K+-ATPase activity of gM1/2+NK beta(1) was inh ibited both by ouabain and Sch-28080. The 50% inhibition concentration for Sch-28080 was 20-60 nM. Sch-28080 at 10 mu M did not inhibit Mg2- and P-i-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+HK 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 parti al 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 chimer a.