臺灣博碩士論文加值系統

Calneuron為CaBP (calcium binding protein) 家族中的一員，主要表現在神經組織中，於N端有兩個能與鈣離子結合的EF-hand motif，其C端並具有一個疏水性結構。之前研究指出CaBP在細胞內鈣離子訊息傳遞路徑中扮演了重要角色，甚至可以直接調控鈣離子電流，而目前對於calneuron在生理上的相關研究卻非常少。電壓依賴性鈣離子通道( Voltage-dependent calcium channel )能調控細胞外鈣離子的流入進而影響鈣離子在細胞內所引發的各種反應。L型鈣離子通道廣泛地表現在各種類型的組織，而P/Q型鈣離子通道則主要表現於神經組織中。本實驗中，我們將大鼠calneuron選殖出來並與L型或P/Q型鈣離子通道同時表現於HEK293t細胞中，發現calneuron可抑制高鉀溶液引發的胞內鈣離子濃度上升。若以電生理技術實驗，結果顯示calneuron可以直接抑制P/Q型鈣離子電流峰值。相較於抑制L型鈣離子電流的大小，calneuron傾向改變開啟L型鈣離子通道的機制。而喪失疏水型C端的calneuron突變型不能發揮calneuron原有功能。由此結果可推測Calneuron能調控胞內鈣離子的訊息傳遞，進而影響胞吐作用與神經細胞間訊息傳導。

Calneuron belongs to calcium binding protein family and is predominantly expressed in neurons. Calneuron has two N-terminal functional EF-hand motifs and a hydrophobic C-terminus. When co-expressed with P/Q-type or L-type Ca(2+) channel in HEK293t cells, calneuron inhibited the intracellular Ca(2+) responses stimulated by high K(+) solution. The P/Q-type Ca(2+) currents measured by whole-cell patch-clamp were inhibited by calneuron; on the other hand, the opening kinetics of L-type Ca(2+) channels was modulated by calneuron. Calneuron mutants with the deletion of their hydrophobic C-terminus did not have the same effect as calneuron in modulating these Ca(2+) currents. These results suggested that calneuron may modulate intracellular Ca(2+) signaling pathway and participate in neuronal function by interacting with the Ca(2+) channels

目次
口試委員審定書
誌謝 i
縮寫表 ii
中文摘要 iv
英文摘要 v
1 前言
1.1 鈣離子的重要性 1
1.2 L型及P/Q型鈣離子通道 1
1.3 鈣離子結合蛋白家族 3
1.4 實驗目標 5
2 材料與方法
2.1 化學藥品 7
2.2 溶液 7
2.3 分子生物技術 8
2.4 細胞培養繼代 9
2.5 細胞轉染 9
2.6 螢光鈣離子顯影 9
2.7 電生理 10
2.8 統計 11
3 結果
3.1 Calneuron 和CaBPs序列有高度相對應性 12
3.2 Calneuron 具有2個EF-hand motif及一個疏水性C端 12
3.3 Calneuron 能抑制高鉀溶液引發的胞內鈣離子濃度上升 13
3.4 Calneuron 抑制了P/Q型鈣離子電流 14
3.5 Calneuron 影響了L型鈣離子通道的啟動機制 14
3.6 去除C端的calneuron突變型影響了其抑制功能 16
4 討論 18
5 參考文獻 22
6 表 29
7 圖 30

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