The Na+ and K+-activated adenosine triphosphatase (Na+K- ATPase) is an intrinsic membrane protein which transports Na+ and K+ across the plasma membrane against electrochemical gradients. This is essential for maintenance of low (Na+) and high (K+) inside the cell. The resulting gradients underly many important physiological processes, including conduction of electrical stimuli in nerve, excitation of muscle, co-transport of sugars and amino acids, counter-transport of ions, maintenance of osmotic pressure, and regulation of cell volume. Our laboratory will address several important, but poorly understood aspects of the (Na+K)-ATPase: (1) structure-function relationships, (2) genomic structure, and (3) molecular mechanism of the regulation of biosynthesis in a developmental system. These studies will take advantage of unique properties of the (Na+K)-ATPase of brine shrimp. Brine shrimp contain two forms of alpha-subunits of the (Na+K)-ATPase which have different activities. Structure- function relationships will be examined by determining the properties of chimeric alpha-subunits in which key domains of one form of alpha-subunit have been substituted by those from the other. The modification of primary structures will be accomplished by recombinant DNA technology. Recombinant DNA technology will also be used to prepare partial-length subunits in order to identify those domains which are required for protein translocation. The ability of these partial length and/or chimeric proteins to effect insertion into the membrane will be examined in cell-free translation systems. This system will also be used to test for involvement of signal recognition particle. A brine shrimp genomic library will be constructed in order to examine the genomic structure of the genes encoding the subunits. The genomic clones will provide tools for future experiments concerning the regulation of gene expression and answer questions regarding the evolution and linkage of the genes encoding the subunits. Portions of the genomic sequence which may be involved in regulation will be identified by gel retardation assays and DNase footprinting. The sequences of these areas will also be determined. The levels of mRNA alpha and mRNA beta increase dramatically during the first 24 hr of development of the brine shrimp. Molecular mechanisms of regulation of the levels of these mRNAs will be examined, including alterations in transcription, processing, or recruitment from RNP.
