| Two delayed rectifier K+ currents have been documented in porcine granulosa cells (PGC): IKs and IKur . This research focuses on their function and modulation.; Previous work from our lab determined that blocking various components of these K+ channels influences PGC physiology differently. Here, 4-aminopyridine (4-AP), a non-specific KCNA channel (the molecular basis for IKur current) antagonist, was used to investigate the function of total IKur in PGC. Progesterone production of cultured PGC was decreased by 4-AP, without changing cell proliferation. This effect is due to complex interactions of various factors: 4-AP decreases cAMP production; down-regulates key steps of steroidogenic pathway, including the expression of steroidogenic acute regulatory protein (StAR) and the production of estrodiol, an amplifier of steroidogenesis. The 4-AP-mediated decrease in progesterone production results from increased intracellular K+ concentration, cell size and intracellular Cl− concentration. Thus, granulosa cell IKur appears to be important for maintenance of cell volume, ionic gradients and steroid synthesis.; The IKs channel can be modulated by different protein kinases. The cAMP-activated protein kinase (PKA) enhances IKs in various mammalian myocytes, but decreases it in granulosa cells. Here, possible reasons for this tissue specific channel regulation have been investigated. Pharmacological experiment suggests that IKs channel stoichiometry in PGC is different from that of cardiomyocyte. Results from molecular biology and protein chemistry experiments show differential expression of PKA targeting proteins (A&barbelow;-k&barbelow;inase a&barbelow;nchoring p&barbelow;rotein, AKAP) in PGC and myocardium. Yotiao, the AKAP tethering PKA to IKs channel complex in cardiac myocytes, is absent in PGC. Over expression of IKs channel proteins at a physiologically relevant ratio in a granulosa cell line, PGC-2, reconstitutes the phenotypes of PKA effect on IKs current in native tissues: PKA activation increases recombinant IKs in the presence of yotiao (myocardiac-like), but decreases the current without yotiao (granulosa-like). These results indicate the importance of AKAP interaction, as well as channel protein stoichiometry in channel regulation.; In summary, this thesis adds to the knowledge about potassium function in granulosa cells, and is the first to provide mechanisms for tissue specific PKA modulation on K+ channels. |
