Molecular and Cellular Studies of Ca2+ Transport ATPase

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DESCRIPTION (provided by applicant): The Sarco-Endoplasmic Reticulum Ca2+ ATPase (SERCA) is the operator of active Ca2+ transport into intracellular stores. The stored Ca2+ is in turn released to trigger cytosolic Ca2"1" signaling. In cardiac and skeletal muscle Ca2+ transport and Ca2+ signaling play prominent roles in control of relaxation and contraction, as well as other functions such as transcriptional activation. These functions are altered in cardiac failure. The aims of this project are: (1) clarification of the molecular mechanism whereby ATP is utilized to move Ca2+ against a concentration gradient; (2) establishment of strategies for gene transfer into cardiac myocytes, and definition of the consequences of overexpression or silencing SERCA and other genes encoding Ca2+ signaling proteins. The research related to aim (1) will produce specific modifications in native and recombinant ATPase by protein chemistry and site directed mutagenesis, and will define the effects of these modifications on the sequential ATPase reactions that are coupled to Ca2+ transport. The findings will be related to crystallographic data and diffraction analysis, to indicate how various ATPase protein domains and specific amino acid residues are involved in energy transduction. Binding sites and specific effects of inhibitors will be defined. It is expected that the mechanism of this enzyme, as a prototype of active transport and energy transduction, will be solved at the molecular and atomic level. The research related to aim (2) will be mostly based on exogenous cDNA delivery to cardiac myocytes by means of recombinant adenovirus vectors under control of specific promoters, thereby optimizing gene transfer and silencing strategies for basic studies of cardiac cell physiology in culture. The functional consequences of SERCA up- or downregulation on Ca2+ signaling, contraction/relaxation cycle and cellular homeostasis will be defined. In addition the effects of silencing specifically SERCA or other genes (i.e., calcineurin) on transcription and expression of other proteins and remodeling of the Ca2+ signaling pathways will be studied. This work will allow us to explore and clarify a new and important concept indicating that in addition to short term functional modulation (i.e., adrenergic), long term changes in copy number, diversity and profile of Ca2+ signaling proteins are important factors in cardiac remodeling, hypertrophy, failure, and possible treatment.