Related Experiment Videos
Ouabain-resistant mutants of the rat Na,K-ATPase alpha 2 isoform identified by using an episomal expression vector
V Canfield1, J R Emanuel, N Spickofsky
1Roche Institute of Molecular Biology, Roche Research Center, Nutley, New Jersey 07110-1199.
Abstract:
Site-directed mutagenesis was used to identify residues responsible for the greater than 1,000-fold difference in ouabain sensitivity between the rat Na,K-ATPase alpha 1 and alpha 2 isoforms. A series of mutagenized cDNAs was constructed that replaced residues of the rat alpha 2 subunit with the corresponding residues from the rat alpha 1 subunit. These cDNAs were cloned into a mammalian episomal expression vector (EBOpLPP) and expressed in ouabain-sensitive primate cells. Either of two single substitutions introduced into the rat alpha 2 subunit cDNA (Leu-111----Arg or Asn-122----Asp) conferred partial resistance (approximately 10 microM ouabain) upon transformed cells. This resistance was intermediate between the levels conferred by the rat alpha 1 cDNA (approximately 500 microM ouabain) and the rat alpha 2 cDNA (approximately 0.2 microM ouabain). A double substitution of the rat alpha 2 cDNA (Leu-111----Arg and Asn-122----Asp) conferred a resistance level equivalent to that obtained with rat alpha 1. These results demonstrate that the residues responsible for isoform-specific differences in ouabain sensitivity are located at the end of the H1-H2 extracellular domain. The combination of site-directed mutagenesis and episomal expression provides a useful system for the selection and analysis of mutants.
Insights
Site-directed mutagenesis revealed specific amino acid residues in rat Na,K-ATPase isoforms that dictate ouabain sensitivity. These key residues are located in the H1-H2 extracellular domain, impacting drug interactions.
Area of Science:
- Biochemistry
- Molecular Biology
- Pharmacology
Background:
- The Na,K-ATPase (sodium-potassium adenosine triphosphatase) is a crucial ion pump with distinct isoforms.
- Significant differences in ouabain sensitivity exist between rat Na,K-ATPase alpha 1 and alpha 2 isoforms, suggesting unique structural determinants.
- Understanding these differences is vital for comprehending drug interactions and cellular ion homeostasis.
Purpose of the Study:
- To pinpoint the specific amino acid residues responsible for the >1,000-fold ouabain sensitivity difference between rat Na,K-ATPase alpha 1 and alpha 2 isoforms.
- To investigate the functional impact of substitutions within the H1-H2 extracellular domain on ouabain binding.
- To establish a robust system for analyzing Na,K-ATPase mutants.
Main Methods:
- Utilized site-directed mutagenesis to create modified rat Na,K-ATPase alpha 2 subunit cDNAs.
- Replaced specific residues in the alpha 2 subunit with corresponding residues from the alpha 1 subunit.
- Cloned mutagenized cDNAs into an episomal expression vector (EBOpLPP) for expression in ouabain-sensitive primate cells.
Main Results:
- Single amino acid substitutions (Leu-111 to Arg or Asn-122 to Asp) in the rat alpha 2 subunit conferred intermediate ouabain resistance (~10 µM).
- The wild-type rat alpha 1 isoform showed high resistance (~500 µM ouabain), while the wild-type alpha 2 isoform exhibited sensitivity (~0.2 µM ouabain).
- A double substitution (Leu-111 to Arg and Asn-122 to Asp) in the alpha 2 subunit mimicked the ouabain resistance of the alpha 1 isoform.
Conclusions:
- The identified residues (Leu-111 and Asn-122) at the H1-H2 extracellular domain terminus are critical for isoform-specific ouabain sensitivity.
- Site-directed mutagenesis combined with episomal expression offers an effective platform for mutant selection and analysis of Na,K-ATPase.
- These findings elucidate the molecular basis of differential ouabain interaction with Na,K-ATPase isoforms.