臺灣博碩士論文加值系統

N型電壓依賴性鈣離子通道 (Cav2.2)受到細胞膜電位上升而開啟，當鈣離子流入細胞內，會產生負回饋作用抑制通道活性。鈣調素 (Calmodulin)有四個EF-hand的鈣離子結合位，可以調控多種通道活性。Calneuron I (CalnI)與鈣調素同鈣離子結合蛋白 (CaBP)家族有高度同源性。CalnI會調控囊泡運輸 (vesicle trafficking)從高基式體至細胞膜，之前研究也指出CalnI會抑制表現在293T細胞中Cav2.2的電流，然而CalnI和Cav2.2之間的關聯性仍然不清楚。Cav2.2於C端一段IQ motif被認為是鈣調素的結合位；我們猜測也可能為CalnI的結合位置，將Cav2.2的IQ motif突變無法受鈣調素調控，利用電生理紀錄Cav2.2 IQ motif突變和CalnI共同表現在293T細胞中的電流變化；我們的結果發現CalnI會抑制Cav2.2的電流無論IQ motif是否有突變。之前研究指出CaMKIIβ與CalnI在酵母雙雜交 (Yeast Two-Hybrid)有交互作用， CaMKIIβ也會被鈣調素活化，參與磷酸化反應與調控鈣離子通道，因此CalnI也有可能藉由調控CaMKIIβ來調控Cav2.2，藉由GST- CalnI作誘餌蛋白與表現在細胞中的CaMKIIβ做pull down處理，並利用西方墨點法觀察兩個蛋白是有交互關係。相反的，GST-CalnI和CaMKIIα的結合效率並不高。從我們的實驗結果認為CalnI不是藉由IQ motif抑制Cav2.2的電流；由於CaMKIIβ會影響突觸可塑性，並且在CalnI和CaMKIIβ的交互關係顯示，CalnI有可能在突觸可塑性中扮演部分角色。

N-type voltage gated Ca2+ channel (Cav2.2) opens when membrane potential elevates, then the channels will be inactivated by the influxed of Ca2+. Calmodulin (CaM), a protein with four EF-hand Ca2+-binding motifs, is involved in regulating versatile channel activities. Calneuron I (CalnI) is homologous to the calmodulin and belongs to the Ca2+ binding protein family. CalnI is known to control the vesicle trafficking from Golgi to the plasma membrane and our previous results shows that CalnI suppresses the currents of Cav2.2 expressed in 293T cells. However, it is not clear CalnI interacts with Cav2.2. CalnI and Cav2.2 mutations in the IQ motif were co-expressed in 293T cells to examine the effects of CalnI on Ca2+ currents using whole-cell patch clamp technique. The IQ motif at the C-terminal of Cav2.2 is proposed to be the CaM binding site; it is possible that CalnI binds to this motif. Our results showed that CalnI suppressed the Cav2.2 current density with or without mutations in the IQ motif. Using yeast-two-hybrid, my lab has previously shown that CalnI interacts with CaMKIIβ. To confirm this interaction, I used GST-CalnI as the bait and the CaMKIIβ expressed in 293T cells could be pull down. In contrast, GST-CalnI did not pulldown CaMKIIα efficiently. Our results suggest that CalnI may not interact with the Cav2.2 through the conserved IQ motif to suppress the Ca2+ current. By interacting with CaMKIIβ, CalnI may be involved in regulating the syanptic plasticity.

致謝……………………………………………….…………………………………….i
摘要……………………………………………….…………………………………...ii
英文摘要………………………………………………………………………..……..iii
1. 緒論………………………………………………………………………..……….1
1.1 鈣離子的重要性………………...................1
1.2 電壓依賴鈣離子通道…………………….……………….…………….1
1.3 N型電壓依賴鈣離子通道………………………………….…………..2
1.4 鈣離子結合蛋白……………………………………………..……………3
1.5 Calneuron………………………………………………………………….4
1.6 CaMKII……………………………………………………………………5
2. 實驗目標…………………………………………………………..………………6
3. 實驗設計………………………………………………………….……………….6
4. 材料與方法……………………………………………………………….……….7
4.1 化學藥品………………………………………………………………….7
4.2 溶液…………………………………………………………….…………7
4.3 細胞培養………………………………………………………….………9
4.4 蛋白質純化及染色………………………………………………………11
4.5 電生理紀錄………………………………………………………………12
5. 結果……………………………………………………………………….………14
5.1 Cav2.2的電流-電位關係…………………………………………...……14
5.2 Cav2.2與Cav2.2 IQ-motif突變株的電流-電位關係比較…………..…14
5.3 CalnI抑制Cav2.2的電流………………………………………..………15
5.4 CalnI抑制Cav2.2 IQ-motif突變株的電流………………………….…16
5.5 CalnI抑制Cav2.2 IQ-motif突變株的電流不需要鈣離子的參與….…17
5.6 CalnI需C端疏水端才能有效抑制Cav2.2 IQ-motif突變株的電流…..18
5.7 CalnI和CaMKIIβ的交互關係…………………………………………19
5.8 CalnI和CaMKIIα的交互關係…………………………………………20
6. 討論…………………………………………………………………….…………21
6.1 CalnI 對Cav2.2的調控…………………………………………………21
6.2 CalnI對Cav2.2的生理意義……………………………………..………22
6.3 CalnI與CaMKII的作用……………………………………..…………23
7. 參考文獻……………………………………………………………….…………24
8. 圖…………………………………………………………………….……………32

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