Lois K Lane

The specific aims ofthis proposal are to identify amino residues that are important for enzymefunction in 1 the H7h8 extracellular alpha subunit domain, which ispostulated to be involved in both NaK and cardiac glycoside interaction; 2 the highly chargedNH2-terminal alpha domain, which appears to beinvolved in K= deocclusion; and 3 to identify domains and residues thatare involved directly and indirectly in the enzyme's interaction withouabain and related drugs. The experimental approach for Aims 1 & 2 is to use site-directedmutagenesis and expression of the ouabain-insensitive rat alpha1 isozyme inHeLa cells, which contain an endogenous ouabain-sensitive isoform of NaK- ATPase. Selection of stable clones with either ouabain or G418 enables usto quicklydetermine if any mutant enzyme is capable of catalyzing NaKtransport or if it is 'lethal'. Active mutant enzymes will be functionallycharacterized with respect to Nak-ATPase:E-P ratio. I50 for ouabain, 86Rbuptake, and K05's for ATP, Na and K The ability of inactive mutantenzymes selected with G418 to be phosphorylated from ATP and Pi will alsobe measured. In addition, some mutant cDNA cassettes will be cloned intoa modified rat alpha 1S cDNA, that encodes a ouabain-sensitive Na,K-ATPaseKD=1.57 nM, and expressed in ouabain-insensitive NIH3T3 cells. Thisallows 3H ouabain binding to be used as a tool for measuring the affinityof the mutant enzymes for ouabain, as well as the enzyme's affinity forthose ligands that regulate ouabain binding eg, ATP. Pi, vanadate.Na, K and Mg The comparison of functional parameters between wild type and mutantenzymes will define the roles that alpha subunit domains and specificresidues play in th function of the NaK-ATPase. These studies arecurrently ongoing in the applicant'slaboratory. Good progress on specificaims 1 and 2 has already been achieved and initial studies on aim 3 areunderway.

Grant: #5-R01-HL-49204-05-A0-S0-E0 Investigators:Lane, Lois 04-01-1995 -02-28-2002 National Heart, Lung and Blood Institute Structure-Function Study of Na, K-ATPase Role:PI $1,409,368.00 Closed Level:Federal

Ligand,Mutant,Ouabain,Catalyst,Rubidium,Hela Cell,Transfection,Phosphorylation,Protein Folding,Chemical Binding,Enzyme Mechanism,Protein Sequence,Western Blotting,Complementary Dna,Molecular Cloning,Northern Blotting;,Synthetic Nucleic Acid,Intracellular Transport,Sodium Potassium Atpase,Site Directed Mutagenesis,Protein Structure Function,Animal Genetic Material Tag