Identification of a region within the Na,K-ATPase alpha subunit that contributes to differential ouabain sensitivity.

Abstract

To analyze determinants within the Na,K-ATPase a subunit that contribute to differential ouabain sensitivity, we constructed and expressed a panel of chimeric cDNA molecules between ouabain-resistant and ouabain-sensitive a subunit cDNAs. When introduced into ouabain-sensitive monkey CV-1 cells, ouabainresistant rat al subunit cDNA and chimeras in which the 5' end of ouabain-sensitive human al or rat OL2 subunit cDNA was replaced by the 5' end of rat al subunit cDNA conferred resistance to 100 ,uM ouabain. Monkey cells transfected with the reciprocal chimeras were unable to survive selection in 1 ,uM ouabain. Rat a2 subunit cDNA and a chimera in which the 5' end of rat al subunit cDNA was replaced by the 5' end of rat a2 subunit cDNA conferred resistance to 0.5 ,uM ouabain. These results suggest that determinants of ouabain resistance reside within the amino-terminal portions of the rat otl and at2 subunits. Expression of chimeric a subunit cDNAs should prove useful for elucidating the structural basis of Na,K-ATPase function.

High-affinity ouabain binding by a chimeric gastric H + ,K + -ATPase containing transmembrane hairpins M3-M4 and M5-M6 of the α 1 -subunit of rat Na + ,K + -ATPase

“…The presence of these polar uncharged amino acids in the M1͞M2 loop thus explains the high affinity for ouabain in chimera HN34͞56. It has to be mentioned that although the first mutagenesis studies suggested a primary role of the M1-M2 hairpin in ouabain binding to Na ϩ ,K ϩ -ATPase (7,12,13,31), later studies indicated that other parts of the enzyme are also involved in the interaction with ouabain (9-11, 15, 18-20, 32, 33). It has been suggested that the overall conformation of the first extracellular loop may influence ouabain sensitivity indirectly by altering the stability or structure of the intermediate of the Na ϩ ,K ϩ -ATPase catalytic cycle that is competent to bind ouabain.…”

“…It is now generally accepted that amino acids present in the first extracellular loop of Na ϩ ,K ϩ -ATPase are involved in the binding of ouabain (7)(8)(9)(10)(11). The border residues of this loop, Arg113 and Asp124, are responsible for the ouabain-resistant character of the rat enzyme (12,13).…”

Ouabain-resistant mutants of the rat Na,K-ATPase alpha 2 isoform identified by using an episomal expression vector.

“…To assess the level of ouabain resistance derived from (4). The EBOpLPP expression system thus allows rapid and efficient analysis of the ouabain sensitivity of Na,K-ATPase a-subunit cDNAs.…”

“…The three rodent ax-subunit isoforms differ -1,000-fold with respect to ouabain sensitivity. Cell lines transfected with rat a2 or a3 cDNAs express a ouabain-sensitive Na,K-ATPase, whereas cell lines transfected with rat or mouse atl cDNAs express a ouabain-resistant Na,K-ATPase (4,5,11,15). However, the molecular basis for differential ouabain sensitivity among a-subunit isoforms has not yet been elucidated.…”

“…An initial series of experiments identified a region within the a subunit involved in differential ouabain sensitivity. Analysis of chimeras containing different segments of ouabain-resistant and ouabain-sensitive a-subunit cDNAs mapped a region responsible for ouabain resistance to the amino-terminal 330 residues of rat al subunit (4). We have used site-directed mutagenesis and an episomal expression vector (EBOpLPP) to identify the specific residues responsible for differences in ouabain sensitivity of the rat al and a2 isoforms.…”