牛腎上腺嗜鉻細胞中NCS-1維持了正常的胞吐活動

Abstract

鈣離子結合蛋白的家族種類眾多，其中包含具有EF hands結構，可與鈣離子結合的鈣離子感應蛋白，如neuronal calcium semsor-1 (NCS-1)，它的結構含有N端myristoylation及三個有功能的EF hands。前人的研究指出，NCS-1會影響分泌膜囊的回收再利用，但詳細機制尚未釐清。我們在牛腎上腺髓質細胞中表現NCS-1，運用安培法測量分泌出的神經傳遞物質以及影像技術來偵測胞內鈣離子變化，以研究高鉀刺激所造成突觸小泡釋放的機轉。我們發現，相較於對照組，表現NCS-1 的細胞，其胞吐作用沒有顯著變化；表現NCS-1G2A (無法結合細胞膜)或NCS-1R102Q (發現在某些自閉症患者)的細胞，其胞吐程度降低，氧化電流和電量均變小，顯示有抑制胞吐作用的現象及突觸小泡內的傳導物質可能減少；而NCS-1E120Q (無法結合鈣離子) 則增加胞吐電量。胞内鈣離子濃度變化的結果顯示，過度表現NCS-1G2A的細胞有顯著較低的鈣離子反應，而表現NCS-1與其他變異的實驗組，則與控制組細胞相近。這些結果顯示，調節胞吐作用的功能與結合到膜上能力有很大相關；而失去鈣離子結合能力，並不影響NCS-1調節的作用。因此NCS-1可能參與在調控分泌膜囊的回收再利用機轉中，並在神經突觸的長期變化，扮演重要的角色。

Many proteins have a Ca2+ binding capability and involved in various physiological activities. Calcium sensor protein family belongs to the calcium binding protein group and has EF hands for Ca2+ binding. Neuronal calcium sensor-1 (NCS-1) has an N-terminal myristoylation site, one cryptic EF hand and 3 functional EF hands. NCS-1 overexpression affects the vesicle recycling, however, the mechanism is not clear. In this report, we monitor the catecholamine release and [Ca2+]i evoked by high K+ depolarization to characterize the roles of NCS-1 in the stimulus-secretion coupling in bovine chromaffin cells. We found that cells overexpressing NCS-1 had similar exocytosis level as the control group expressing GFP; cells overexpressing NCS-1G2A (no plasma membrane anchoring capability) or NCS-1R102Q (identified in autism patients) had less exocytosis events, and smaller oxidation currents; in contrast, cells overexpressing NCS-1E120Q (loses Ca2+-binding ability) increased the exocytosis level. For the intracellular Ca2+ response, all constructs caused similar responses as the control group except NCS-1G2A which had a smaller elevation than the control group. These results indicated that the plasma membrane targeting capability is important for the regulatory role of the NCS-1 in the stimulus-secretion coupling; however, losing the Ca2+-binding ability does not affect the regulatory role of the NCS-1. In summary, NCS-1 may be involved in regulating the synaptic vesicle recycling and playing an important role in long term synapse plasticity changes.