The sodium/potassium adenosine triphosphatase (Na+,K+-ATPase) is the membrane protein that maintains the cell's Na+/K+ electrochemical gradient via the active transport of Na+ and K+ across the plasma membrane. Through coupling with other cation transport mechanisms, it is involved in the regulation of cell volume, differentiation and proliferation; ion/solute uptake in the kidney, liver, intestine; propagation of action potential of nerve, skeletal and cardiac muscles, modulation of synaptic action, and cardiac glycoside inotropy. It has also been implicated in cardiovascular hypertension, cardiac hypertrophy, brain edema, cystic fibrosis, and autohemolytic red cell membrane diseases. Its study is, therefore, important in basic and medical sciences. Molecular biology coupled with cell and protein biochemistry provides an incisive tool to the analysis of structure-function relationships and gene regulation of the Na+,K+-ATPase alpha- subunit. Recently we have characterized cDNA clones coding for three rat alpha-subunit isoforms alpha 1, alpha 2, and alpha 3). They are encoded by a multigene family that is differentially expressed in a tissue-specific and developmental manner. Of interest is their unique developmental expression in the heart. The alpha 1 isoform is constitutive; alpha 2 is feta/neonatal predominant; and alpha 3 is adult predominant. We have obtained the complete primary structure of alpha 1 and alpha 2 isoforms. This research proposes to study the molecular genetics of these cardiac Na+,K=-ATPase alpha-subunit isoforms, thus gaining insight into their role(s) in the heart during development and in pathology by aiming to: 1) complete the structural analysis of alpha 3 determining its complete primary structure, 2) establish a human tissue culture system to study the synthesis of the rat alpha-subunit isoforms, 3) define the functional differences of the presently isolated alpha 1, alpha 2, and alpha 3 isoforms, 4) assess the structure-functional significance of the putative ouabain- binding sites I and II via site-directed mutagenesis, and 5) study the Na+-pump's role(s) in cardiac development and cardiac hypertrophy by assessing the modulation of expression of the Na+- pump isoforms in rat by Northern blot, in-situ hybridization, and S1 nuclease mapping analyses. This research study will provide insight into the mechanisms of the Na+-pump's general and highly specialized functions. It will also pave the way for future research on precise mechanisms of Na+-,K+-ATPase structure- function relationships and gene regulation in cardiac development, hypertrophy, and cardiac-glycoside induced inotropy.
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