Expression of an ouabain-resistant Na,K-ATPase in CV-1 cells after transfection with a cDNA encoding the rat Na,K-ATPase alpha 1 subunit.
We have used a gene transfer system to investigate the relationship between expression of the rat Na,K-ATPase alpha 1 subunit gene and ouabain-resistant Na,K-ATPase activity. A cDNA clone encoding the entire rat Na,K-ATPase alpha 1 subunit was inserted into the expression vector pSV2neo. This construct (pSV2 alpha 1) conferred resistance to 100 microM ouabain to ouabain-sensitive CV-1 cells. Hybridization analysis of transfected clones revealed the presence of both rat-specific and endogenous Na,K-ATPase alpha 1 subunit DNA and mRNA sequences. A single form of highly ouabain-sensitive 86Rb+ uptake was detected in CV-1 cells, whereas two distinct classes of ouabain-inhibitable uptake were observed in transfectants. One class exhibited the high ouabain sensitivity of the endogenous monkey Na,K-ATPase, while the second class showed the reduced ouabain sensitivity characteristic of the rodent renal Na,K-ATPase. Examination of the ouabain-sensitive, sodium-dependent ATPase activity of the transfectants also revealed a low affinity component of Na,K-ATPase activity characteristic of the rodent kidney enzyme. These results suggest that expression of the rat alpha 1 subunit gene is directly responsible for ouabain-resistant Na,K-ATPase activity in transfected CV-1 cells.
Emanuel
J R
JR
Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06510.
Schulz
J
J
Zhou
X M
XM
Kent
R B
RB
Housman
D
D
Cantley
L
L
Levenson
R
R
D000465:Algorithms; D000818:Animals; D004247:DNA / Q000032:analysis*; D005786:Gene Expression Regulation / Q000187:drug effects*; D007700:Kinetics; D010042:Ouabain / Q000494:pharmacology*; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D012413:Rubidium / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000235:genetics*; D013327:Strophanthidin / Q000032:analysis; D014162:Transfection
D012333:RNA, Messenger; D010042:Ouabain; D013327:Strophanthidin; D004247:DNA; D000254:Sodium-Potassium-Exchanging ATPase; D012413:Rubidium