The long term objectives of this study are to understand the biochemical properties of the Na,K-ATPase, describe the functional role of the alpha isoforms and to define the regulation of the genes coding for the various subunits of this enzyme. During this granting period, the principal investigator proposes to (1) Define the enzymatic and functional characteristics of the alpha 4 isoform which they have cloned. This will be accomplished by expressing this isoform in yeast and determining its apparent Na+ and K+ affinities as well as other properties of the enzyme. 2) Determine the basis for the differential function of the alpha 1 and alpha 2 isoforms. In previous studies using gene targeting it has been shown that these two isoforms play a differential role in heart contractility. Using chimeras between these two isoforms, the basis for this difference will be defined. It is possible that the alpha 1 and alpha 2 isoforms are located in different regions of the plasma membrane and that the location is responsible for the differences observed. 3) Analysis of the skeletal muscle function in mice lacking one copy of either the alpha 1 and alpha 2 subunit genes of the Na,K ATPase. While the Dr. Lingrel's group has shown that animals lacking one copy of either the alpha 1 or alpha 2 isoform show characteristic changes in heart function, it is not yet known whether other tissues such as the muscle are also affected. Contractility in extensor digit rum longs and soleus muscles will therefore be examined. 4) The regulator elements in the alpha 2 isoform gene which are responsible for tissue specific expression will be defined. This isoform is essentially in skeletal muscle, adult heart, and brain. Dr. Lingrel's group has already demonstrated that 1,565 bases upstream of this gene drive correct expression in transgenic animals. The cis elements required for expression and the identity of the trans acting factors that interact with these sites will be established.
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