Na,K-ATPase, also known as the sodium pump, is an integral membrane protein found in all animal cells. This enzyme maintains electrochemical gradients through the exchange of intracellular Na+ for extracellular K+ coupled to ATP hydrolysis. In the heart, the sodium pump maintains ion gradients requisite for electrical excitability, and is the contractility. The overall aim of this proposal is to characterize the structure and regulation of cardiac Na,K-ATPase. Structure studies will be conducted on dog heart Na,K-ATPase where we have evidence for multiple forms of the catalytic subunit (alpha and alpha+). Full length cDNAs to cardiac alpha, alpha+ and beta will be isolated from a cDNA library using synthetic oligonucleotides, sequenced, and protein structure predicted using existing algorithms. The full length cDNAs (in plasmid vectors) will be expressed in yeast and mammalian cells to test the hypothesis that beta subunit is unnecessary for Na,K-ATPase assembly or activity. The expression systems will also be used to address structure-function relationships in putative critical regions of alpha by site-directed mutagenesis of the cDNA. Regulation studies will be conducted on primary cultures of rat heart cells. We will establish whether the upregulation observed in heart in response to incubation in low K+ or in response to thyroid hormone is due to increased synthesis of pump subunits by assaying subunit mRNA levels with cDNAs, and will define the ionic signals that trigger the response to low K+ by independently regulating Na+, K+, and H+. We will determine whether regulation is transcriptional using nuclear run- off assays, and determine the signal (ton/hormone) sensitive regulatory regions of genes, the ultimate site of gene regulation, by transfecting hybrid genes and assaying for the ability of putative regulatory sequences to confer signal sensitivity to a heterologous gene. Accomplishing these aims will contribute towards an explanation of the mechanics of differential function and regulation of Na,K-ATPase in heart compared to other tissues and may potentially impact the understanding of abnormal cardiac function in disease states.
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