Targeting the Mitochondrial Calcium Uniporter, Phase I

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DESCRIPTION (provided by applicant): The long-range goal of this project is to obtain an understanding of mitochondrial Ca2+ homeostasis at the molecular level. The Ca2+ uniporter is a key player in that regard, in that it is a central member of the elaborate system which transports Ca2+ into and out of mitochondria. The uniporter is therefore involved in maintaining intracellular Ca2+ homeostasis, in regulating energy production, in pathological processes that lead to necrotic cell death, and in cell death occurring via apoptosis. These considerations have established the uniporter as an interesting membrane protein from the perspective of basic research, and as attractive target for drug development. Despite this, there is no information about the molecular properties of the uniporter, while it is clear that such information will be required to make significant progress from either perspective. This program initiates molecular studies of the uniporter by undertaking to clone it from multiple sources and to express the proteins in yeast mitochondria, which lack an endogenous analog. Several novel strategies are employed. In one, cDNA libraries are expressed in a yeast cell line that has been engineered to express the photoprotein aequorin in mitochondria. Light emission is then used to distinguish between cells that also express the uniporter and those that do not. In another strategy, libraries are expressed in mutant yeast that grow poorly at normal Ca2+ levels because intracellular storage volumes cannot be utilized. These are replaced by the mitochondrial matrix space in cells the express the uniporter, allowing their identification by rate of growth. A third strategy uses amino acid sequence information derived from an apparent section of uniporter structure to design primers and to clone it by PCR. A fourth strategy uses bioinformatics to search genomic databases for uniporter candidates, based upon a set of motifs that should be included according to existing data. In all cases, activities identified and expressed in yeast undergo a detailed examination, comparing their bioenergetic characteristics to those of the authentic uniporter.