Identification of a region within the Na,K-ATPase alpha subunit that contributes to differential ouabain sensitivity.
To analyze determinants within the Na,K-ATPase alpha subunit that contribute to differential ouabain sensitivity, we constructed and expressed a panel of chimeric cDNA molecules between ouabain-resistant and ouabain-sensitive alpha subunit cDNAs. When introduced into ouabain-sensitive monkey CV-1 cells, ouabain-resistant rat alpha 1 subunit cDNA and chimeras in which the 5' end of ouabain-sensitive human alpha 1 or rat alpha 2 subunit cDNA was replaced by the 5' end of rat alpha 1 subunit cDNA conferred resistance to 100 microM ouabain. Monkey cells transfected with the reciprocal chimeras were unable to survive selection in 1 microM ouabain. Rat alpha 2 subunit cDNA and a chimera in which the 5' end of rat alpha 1 subunit cDNA was replaced by the 5' end of rat alpha 2 subunit cDNA conferred resistance to 0.5 microM ouabain. These results suggest that determinants of ouabain resistance reside within the amino-terminal portions of the rat alpha 1 and alpha 2 subunits. Expression of chimeric alpha subunit cDNAs should prove useful for elucidating the structural basis of Na,K-ATPase function.
Emanuel
J R
JR
Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06510.
Graw
S
S
Housman
D
D
Levenson
R
R
D000818:Animals; D001665:Binding Sites; D002678:Chimera; D004247:DNA / Q000235:genetics; D006801:Humans; D010042:Ouabain / Q000494:pharmacology*; D011487:Protein Conformation; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism
D011994:Recombinant Proteins; D010042:Ouabain; D004247:DNA; D000254:Sodium-Potassium-Exchanging ATPase