The work proposed in this project is aimed at attaining a better understanding of the molecular mechanism of the cardiac sarcoplasmic reticulum (SR) Ca2+-ATPase. Calcium ions play a pivotal role in the contraction-relaxation cycle in the heart, and therefore the control and regulation of intracellular (Ca2+] is a crucial factor of cardiac function. The SR plays the primary role in the regulation of cytoplasmic Ca2+ and the SR Ca2+-ATPase is the sole protein responsible for active transport of Ca2+ into the SR. Although a large body of data exists on the kinetic aspects of the transport process, little is known concerning the conformational dynamics of energy transduction or the molecular mechanism of ion translocation. The major aim of this proposed work is to study the structure and function of the Ca2+-ATPase in a cell line in which the function of site-directed mutants can be quickly and accurately assessed. Our discovery of a prokaryotic Ca2+-ATPase which is functionally similar to the cardiac SR Ca2+-ATPase provides us with a """"""""made to order"""""""" model system to augment and complement our studies of the cardiac SR Ca2+- ATPase. In the past three years since our discovery of the prokaryotic Ca2+-ATPase, we have characterized transport, hydrolysis, forward, and reverse phosphorylation, and have analyzed both the steady state and single turnover kinetics of its reaction cycle intermediates. This pump has been purified to homogeneity, the gene cloned and most importantly it has been functionally expressed in E. coli which has no endogenous Ca2+- ATPase. The expression of the Ca2+ pump in E. coli now allows i) the quick and accurate assessment of site-directed mutants and chimeric constructs between the SR and prokaryotic pump, ii) revertant analysis of all critical mutants, iii) the enrichment and screening of randomly generated mutants, and iv) the ability to analyze membrane topography and oligomeric structure utilizing techniques which are unavailable or impossible to use with expression in eukaryotic cell lines.
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