| In Chapter I, I give an overview of the Voltage-dependent Calcium Channel (VDCC) and its importance as a key regulator of intracellular calcium. I attempt to provide a thorough explanation of the nomenclature system used to describe the various VDCC subunits, and discuss the structure and interaction of the various subunits of the VDCC. I also provide a summary of their functional effects, what information any mouse models have provided up until this point, and the clinical relevance of the various subunits of the VDCC. I conclude Chapter I with the specific aims underlying the work laid out within this thesis. In Chapter II, I describe my use of co-immunoprecipitation assay to demonstrate a physical association of gamma6 and Cav3.1 in both HEK cells and atrial myocytes, and that the functionally critical domain and motif of gamma6 are not required for gamma6 interaction with Cav3.1. Furthermore, I describe the use of cell surface membrane protein biotinylation, purification, and quantitative western blot to demonstrate that the functional effect gamma6 confers upon Cav3.1 current is not due to a change in the plasma membrane expression of Cav3.1. In Chapter III, I describe my work using cardiomyocytes of adult rats and adenoviral mediated expression to demonstrate the ability of the gamma6 subunit to associate with Cav3.1 in cardiomyocytes as well as cause a marked decrease in T-type calcium current density when gamma6 expression is increased. Finally, in Chapter IV, I describe my attempts at answering the question of where within Cav3.1 the pharmacologically relevant gamma6hTM1 peptide may intereact. The question was addressed through photoaffinity cross-linking of photoreactive gamma6hTM1 peptide analogues to Cav3.1, as well as GST-pulldown of proteolytic peptides of Cav3.1. The results fall short of answering the question due to the unforeseen inability of mass spectroscopy to adequately detect the highly hydrophobic peptides of Cav3.1. |
