NCS-1與Auxilin-1對胞吞/胞吐作用之影響

Abstract

神經傳遞物質以胞吐釋放後，同時也靠著胞吞作用，來以維持神經細胞細胞膜表面積的恆定。Neuron calcium sensor 1 (NCS-1)是EF-hand鈣離子結合蛋白家族的一員，其會與鈣離子結合並調控神經傳遞物質的釋放。此外，本實驗室之前的研究以Yeast-two-hybrid screening發現NCS-1會與Auxilin-1的C端(胺基酸序列為384至910)結合。Auxilin-1分布在神經細胞突觸上，在網格蛋白調控的胞吞機轉中，幫助網格蛋白從胞吞作用進來的小泡上離開。為更了解兩者扮演的角色，將NCS-1以及Auxilin-1基因在腎上腺嗜鉻細胞中大量表現並給予刺激，觀察鈣離子流入量及細胞膜電容變化。結果顯示，失去與鈣離子結合能力之NCS-1E120Q及無法與細胞膜鍵結的NCS-1G2A在連續刺激下，在鈣離子流入量有降低的趨勢，而NCS-1E120Q則使胞吐量顯著下降。而表現Auxilin-1的細胞，並不影響鈣離子流入量及膜電容變化。而失去與Hsc70結合能力的Auxilin-1H874Q，則使鈣離子流入量及胞吐作用上顯著下降。在胞吞能力上，NCS-1與Auxilin-1都有著較慢的速率。這些結果顯示NCS-1與鈣離子的結合，可幫助小泡來到Readily releasable pool (RRP)，以準備釋放，並幫助細胞在刺激結束後，進行胞吞以維持細胞膜的恆定。Auxilin-1會使細胞RRP的量變小，但不影響總釋放量。而NCS-1與Auxilin-1在牛腎上腺嗜鉻細胞中，有共同的表現位置，與之前研究不同的地方在於NCS-1會參與在胞吞作用上，Auxilin-1會參與到胞吐作用上，因此NCS-1與Auxilin-1兩者之間可能有交互作用，共同調控胞吞胞吐作用。

During synaptic cycle in neurotransmitter release, the surface area of cell membrane maintains homeostasis by exo/endocytosis. Neuron calcium sensor 1 (NCS-1), a member of EF-hand calcium-binding protein family, is shown to regulate neurotransmitter release. Our previous results have shown that the half part of C terminus (a.a. 444-970) in rat Auxilin-1 binds to NCS-1 by yeast-two-hybrid screening using NCS-1 as the bait. Auxilin-1 is expressed in synapse, and facilitates clathrin uncoating from clathrin-coated vesicles. To characterize the roles of NCS-1 and Auxilin-1 in exo/endocytosis, the effects of NCS-1 and Auxilin-1 on evoked exo/endocytosis were monitored by the calcium influx and capacitance measurements in bovine chromaffin cells. In confocal images, overexpressed NCS-1 and Auxilin-1 are colocalized in chromaffin cells. Both mutants NCS-1E120Q with the abolished calcium binding ability and NCS-1G2A with the abolished membrane anchoring ability decreased the calcium influx during repetitive stimuli. Furthermore, NCS-1E120Q significantly reduced exocytosis. In contrast, overexpression of human Auxilin-1 had no significant effect on calcium influx and exocytosis; however, Auxilin-1H874Q mutant which loses its ability to interact with Hsc70 significantly decreased the calcium influx and exocytosis. For endocytosis measured by capacitance, both NCS-1 and Auxilin-1 had a slower rate of membrane retrival. These results indicate that calcium binding to NCS-1 helps vesicles recycling to the plasma membrane, and Auxilin-1 takes part in the vesicle recycling pathway, but Auxilin-1H874Q decreases the endocytosis rate. In conclusion, NCS-1 may interact with Auxilin-1 on the half part of C terminus to regulate exo/endocytosis.