| Summary: | 碩士 === 國立臺灣大學 === 腦與心智科學研究所 === 103 === At the presynaptic terminals, calcium triggers the exocytosis mediated by various calcium binding proteins. Neuronal calcium sensor-1 (NCS-1) belongs to EF-hand superfamily of calcium binding proteins and has an N-terminal myristoylation for membrane association. NCS-1 does not only regulate exocytosis through Ca2+ channels but interact with endocyto-sis related protein. However, the detail how NCS-1 affect the vesicle recycling is not clear. Auxilin is a chaperone molecule involved in endocytosis, facilitating uncoating of clathrin and AP2 from endocytosed vesicle. We have previously verified that NCS-1 interacts with auxilin by yeast-two-hybridization. Therefore, we are interested in how NCS-1 and auxilin regulate the exo- and endocytosis using amperometry recording and calcium imaging. Re-petitive high K+ evoked exocytosis was investigated using bovine chromaffin cells express-ing NCS-1 and mutants. Amperometry recording shows that NCS-1 delayed the spike dis-tribution, suggesting that NCS-1 might decrease the size of the readily releasable pool. The prolonged halfwidth infers that NCS-1G2A might increase the fusion period. In addition, NCS-1E120Q diminished the quanta size, indicating NCS-1 might be involved in neurotrans-mitter filling. When NCS-1-EYFP and ECFP-auxilin C-half were coexpressed in PC12 cells, both Fluorescence resonance energy transfer (FRET) and confocal images revealed that YFP intensity dropped upon ATP stimulation, suggesting that NCS-1 and auxilin dissociate dur-ing stimulation. These results demonstrate NCS-1 regulate the kinetics of fusion pore during exocytosis and may interact with auxilin to regulate the vesicle recycling.
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