NCS-1和Auxilin對牛腎上腺嗜鉻細胞胞吐作用的影響

Abstract

在突觸前神經末梢，鈣離子透過結合蛋白啟動胞吐作用釋放神經傳導物質。Neuronal calcium sensor-1 (NCS-1) 含有EF-motif 結合鈣離子和N端荳蔻酸化修飾以嵌入細胞膜，屬於鈣離子結合蛋白的一員。已知NCS-1會調節鈣離子通道或結合胞吞作用相關蛋白，進而調控突觸前囊泡循環，但相關細節尚不清楚。Auxilin為一伴護蛋白，參與在胞吞作用中幫助網格蛋白脫離內吞囊泡表面。本實驗室先前以酵母菌雙雜合系統中證實NCS-1和Auxilin有相互作用，因此在本研究中想了解NCS-1和Auxilin在牛腎上腺嗜鉻細胞中，如何影響囊泡循環。透過安培紀錄法，以高鉀重複刺激後發現，NCS-1延遲胞吐作用發生，推論NCS-1 會減少可立即釋放的囊泡。NCS-1G2A點突變會延長囊泡膜與突觸膜之融合過程。另外，發現NCS-1E120Q點突變可能會降低神經傳導物質填入囊泡之效果。而利用螢光共振轉移和共軛焦顯微鏡影像技術，在NCS-1-EYFP和ECFP-Auxilin C half 共同表現的PC12細胞上，給予ATP刺激時會使EYFP螢光強度暫時下降。推測NCS-1和Auxilin受到ATP刺激會短暫分離後再次結合。本研究主題釐清了NCS-1和Auxilin在囊泡循環中扮演之功能，深入突觸傳遞之分子機制進行探討。

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.