The long-term objectives of this application are to understand the structure and function of calcium pump molecules which reside in intracellular organelles, and to understand how their regulation influences cell physiology in both smooth muscle and non-muscle cells.
The specific aims are as follows: 1. To isolate and characterize all isoforms encoding intracellular calcium pumps, to determine the level of their expression in different tissues and cell lines, and to define their location within the cell. This will be done using techniques of cDNA cloning to define the isoforms, and using quantitative analyses of steady state message levels (i.e. RNase protection assay) for each of the different cDNA clones identified. We will address the question of intracellular location of the different isoforms, particularly with regard to distinct compartments of calcium storage, by employing isoform specific antibodies in conjunction with immunohistochemical and immunoelectron microscopic methods and membrane fractionation procedures. 2. To express cDNA molecules encoding the different Ca-ATPase isoforms in COS-1 cells, and thus to determine various biochemical parameters of function and structure for each. These parameters will include: extent (if any) of glycosylation, localization within the COS-1 cells, quantitation of the turn-over number of the molecules, both for ATP hydrolysis and for calcium transport, the dependence of the transport function upon calcium concentration, upon ATP (and other nucleotide) concentration, and upon pH. The relation of structure to function will be addressed for properties which differ among the isoforms using chimeric and site-directed mutated molecules. 3. To examine the extent to which the pumping properties of Ca-ATPase isoforms are regulated within the cell. The properties of calcium uptake into saponin permeabilized cells will be examined, using inhibitory isoform specific antibodies to assess the contribution of each species to overall uptake, under a variety of conditions of calcium concentration, pH, etc. The effect of ligands such as PMA, cGMP, cAMP on calcium uptake will be monitored. The association of different calcium pump isoforms with distinct intracellular calcium pools will be addressed. The target for the novel tumour promoter, thapsigargin, will also be investigated. These studies will shed light on the mechanisms governing cytoplasmic calcium concentration - the key element controlling a variety of cellular events - and its regulation by hormonal effectors.
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