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  • Book
    Natalia Gomez-Ospina.
    Digital2010
    Abstract : Voltage-gated calcium channels are an important route of calcium entry into cells and are essential for converting electrical activity into biochemical events. In neurons these channels are vital for synaptic vesicle release and have been implicated in almost every activity-dependent process including survival, dendritic arborization, synaptic plasticity, and gene expression. One of the ways in which these channels regulate cellular behavior is by regulating gene expression but the mechanisms that link calcium channels to the transcription machinery are not well understood. In this thesis I show that a C-terminal fragment of CaV1.2, an L-type voltage-gated calcium channel, translocates to the nucleus and regulates transcription. I show that this calcium channel associated transcription regulator (CCAT), binds to a nuclear protein, associates with an endogenous promoter, and regulates the expression of a variety of endogenous genes that are important for the function of neurons and muscle cells. The nuclear localization of CCAT is regulated by changes in intracellular calcium on a time scale of minutes, suggesting that CCAT integrates information about the electrical activity of the cell. Together these findings reveal an entirely unexpected function for a well-characterized calcium channel. This works also addresses the question of how CCAT is generated. I show that CCAT is not released from proteolysis of full-length Cav1.2 channel but is generated from an mRNA that is transcribed from the 3' end of the Cav1.2 gene (CACNA1C). Consistent with this, I find that CCAT expression is independent of full-length channel protein and that exon 46 of the CACNA1C gene contains a promoter whose transcriptional activity is required for the expression of CCAT. Activity at this promoter, and consequently CCAT expression, is regulated spatially and temporally in the brain having highest expression during embryonic stages and in regions of the brain rich in inhibitory neurons. Analysis of 5' transcriptional starts from CACNA1C and Cap Analysis of Gene Expression (CAGE) tags from genome-wide studies show at least two mRNAs one of which encodes CCAT in vivo and a second transcript that is predicted to encode a membrane bound CCAT containing a voltage sensor. These findings reveal an unexpected mechanism by which CCAT is generated in neurons and provide a unique example by which two proteins with distinct biologic functions can be derived from a single gene. Such transcriptional phenomena may be at play in many other genes throughout the genome and has far reaching implications for prediction of gene products and interpretation of phenotypes in gene mutations and knockout studies.