NCS-1及Auxilin-1對神經傳導的影響

Abstract

鈣離子訊號在神經及神經內分泌細胞的神經傳導物質釋放上扮演很重要的角色。鈣離子的流入誘發許多以鈣離子結合蛋白所調控的訊號傳導路徑。Neuronal calcium sensor-1 (NCS-1)，含有四個能與鈣離子結合的EF-hand motifs，且已知可促進果蠅的突觸傳導。本實驗室之前的研究以yeast-two-hybrid screening發現NCS-1會與Auxilin-1的C端結合。Auxilin-1是幫助網格蛋白從胞吞作用進入的小泡上離開的一個蛋白質。為了解NCS-1及Auxilin-1的功能，以全細胞膜電鉗技術測量腎上腺髓質細胞的膜電容。電容值代表細胞表面積大小並可反映胞吞胞吐作用的活性。在三次連續刺激下控制組的胞吐作用程度並無明顯改變。而細胞表現NCS-1及NCS-1R102Q三次刺激平均之胞吐程度與控制組比較分別被抑制到58.58 %及 34.31 %。相似的抑制作用在Auxilin-1也被觀察到 (36.72 %)。 此外，NCS-1R102Q的胞吞速率則顯著被抑制。為確認其突觸傳導的影響，以Glutamate刺激神經細胞利用Ca2+ imaging的技術觀察細胞反應，顯示NCS-1下游細胞螢光強度有顯著的減弱。結果指出，NCS-1與鈣離子結合可幫助突觸小泡的釋放，然而，過度表現NCS-1的細胞可能因競爭胞內鈣離子的緣故導致胞吐作用被抑制。表現Auxilin-1的細胞則可能使突觸小泡聚積而導致胞吐作用被抑制的影響。

Calcium signaling plays an important role on neurotransmitter release in nervous and neuroendocrine cells. Calcium influx triggers many signal transduction pathways mediated by various calcium binding proteins. Neuronal calcium sensor-1 (NCS-1) contains four EF-hand calcium binding motifs, and is known to enhance the synaptic transmission in Drosophila. In our lab, by yeast-two-hybrid screening with NCS-1 as the bait, a rat cDNA clone of auxilin-1 was identified. Auxilin-1 is a protein involved in uncoating the clathrin from the clathrin-coated vesicle. To characterize the functions of NCS-1 and Auxilin-1, bovine chromaffin cells were patch-clamped at perforated whole-cell mode and the membrane capacitance was recorded. The capacitance value is proportional to the surface area of cell and can be used to reflect the activity of exo-endocytosis. During three repetitive stimulations, the exocytosis level was not changed in control cells. For cell expressing NCS-1 or NCS-1R102Q, the average exocytosis levels at three stimulations were significantly inhibited to 58.58 % and 34.31 % comparing to the control respectively. Similar inhibitory effect was also observed in cells expressing Auxilin-1 (36.72 %). The endocytosis rate was significantly inhibited in cells expressing NCS-1R102Q. To verify their effects on synaptic transmission, cultured neurons were stimulated by uncaging glutamate and the responses were monitored by Ca2+ imaging. Cells expressing NCS-1 or NCS-1R102Q had a significant effect on reducing the Ca2+ responses in downstream neurons. These results suggest that NCS-1 binding with calcium helps synaptic vesicle release. But cells overexpressing NCS-1 maybe compete the intracellular calcium leading to exocytosis inhibition. And Auxilin-1 may cause accumulation of clathrin-coated vesicles resulting in the similar inhibitory effect.