| Neuronal calcium sensor-1 (NCS-1) belongs to a family of Ca2+ sensor proteins which is conserved from yeast to humans. Its originally identified homologue frequenin enhances synaptic efficacy at the neuromuscular junction in Drosophila and Xenopus and initial studies in neuroendocrine (PC12) cells also implicate NCS-1 in increasing Ca2+-dependent exocytosis. Its homologue in yeast, frg1, has been shown to be an essential gene, as the null mutant is lethal. Genetic screens have identified a phosphatidylinositol-4-kinase, pi1kp, as a specific suppressor. Frq1 physically interacts with pi1kp and upregulates its activity suggesting a distinct role of frg1 in modulating phosphatidylinositol-dependent signaling pathways.; In this thesis, the role of NCS-1 in synapse formation and in transmitter release was examined in neuroblastoma (NG108)-myocyte co-cultures. Endogenous NCS-1 partially localizes with SNAP-25 and overexpression of NCS-1 in differentiated NG108 cells facilitates synapse formation and increases the quantal content of evoked aceytlcholine release.; Stable PC12 cells overexpressing NCS-1 were used to examine its localization and role in Ca2+ signaling and exocytosis. NCS-1 is found in the light synaptic vesicle fraction showing a parallel distribution with synaptophysin. Both the potency of and magnitude of uridine 5′-triphosphate (UTP) or bradykinin-evoked increases in intracellular Ca2+ concentrations were enhanced in NCS-1 overexpressing cells. This enhancement correlated with increased UTP-stimulated phosphoinositide turnover. In addition, exocytosis evoked by UTP, but not by high K+, was enhanced by NCS-overexpression. These data indicate that NCS-1 regulates phosphatidylinositol turnover leading to enhanced Ca2+ signaling and exocytosis. In conclusion, these results demonstrate a role for NCS-1 in synaptic transmission and transmitter release in mammalian cell lines by regulating phosphatidylinositol-dependent signaling pathways. |
