指導教授：潘建源臺灣大學：生命科學系蔡依廷Tsai, Yi-TingYi-TingTsai2014-11-262018-07-062014-11-262018-07-062014http://ntur.lib.ntu.edu.tw//handle/246246/261678電壓依賴型鈉離子通道 (Navs)在動作電位啟動及傳遞上，扮演重要角色。鈣調素 (calmodulin, CaM)為鈣離子感受蛋白，可與Navs C端的一段高度保守序列，IQ motif結合而調控其活性，鈣調素N端和C端各有兩個EF-hand鈣離子結合位。本實驗中，將Nav1.4表現於293T細胞中，以全細胞膜片箝技術，測量Na+電流。鈣調素使電流密度從-82.4 ± 7.7顯著增加到-127.8 ± 15.6 pA/pF，而無鈣離子結合功能之突變型鈣調素 (CaMtetra)，則不影響電流密度 (-76.5 ± 12.6 pA/pF)。C端無鈣離子結合功能的突變型鈣調素 (CaM34)，可顯著增加電流密度 (-132.5 ± 13.3 pA/pF)，然而N端無鈣離子結合功能的突變型鈣調素 (CaM12) 則無此效果(-73.0 ± 10.4 pA/pF)。提高細胞內液鈣離子濃度至0.2和10 μM，可更進一步增加電流密度。然而鈣調素會縮短通道由不活化再回到可再開啟的時間，從6.0 ± 0.5增加到6.4 ± 0.8 msec。這些結果顯示在生理狀態下，當細胞內鈣離子濃度上升，鈣調素提高Nav1.4的電流密度度，來調節興奮性細胞的活性。Voltage-gated sodium channels (Navs) are essential for the initiation and propagation of action potentials in excitable cells. Calmodulin (CaM) is a calcium sensor protein and can bind to the highly conserved IQ motif at the intracellular C-terminal of Navs to regulate channel activities. Each of the N- and C-lobes in CaM has two EF-hand Ca2+-binding motifs and is known to have distinct effects in modulating channel activities. In this report, we co-expressed CaM and mutations with Nav1.4 to characterize how CaM regulates the channel activities. I expressed these proteins in 293T cells and measured the Na+ currents with patch-clamp technique in whole-cell mode. CaM significantly enhanced the current density from -87.3 ± 8.8 to -137.3 ± 16.5 pA/pF, however, CaM1234, which has no Ca2+ binding capability, had little effect on the current density (-76.5 ± 12.6 pA/pF). CaM34, which has no Ca2+-binding abilities at the C-lobe, significantly enhanced the current density -135.0 ± 15.6 pA/pF; in contrast, CaM12, which loses Ca2+-binding abilities at the N-lobe, did not have the effect in enhancing the current density (-81.2 ± 13.1 pA/pF). Elevating the Ca2+ concentration in the pipette solution to 0.2 and 10 μM further enhanced the current density in the presence of CaM overexpression.CaM co-expression significantly shortened the recovery time of Nav1.4 from 3.3 ± 0.2 to 3.0 ± 0.0 msec. These results demonstrate that, under physiologiucal conditions when the intracellular Ca2+ concentration elevates, CaM activation leads to the enhancement of the Nav1.4 currents to regulate the activities of excitable cells.致謝 1 摘要 2 中文摘要 2 英文摘要 3 前言 4 電壓依賴型鈉離子通道(voltage-gated sodium channel, Navs ) 4 電壓依賴型鈉離子通道的結構 5 電壓依賴型鈉離子通道離子通透性的選擇 6 電壓依賴型鈉離子通道的組織分布 7 鈣調素(Calmodulin, CaM) 7 IQ motif 9 實驗目的 12 實驗材料與方法 13 化學藥品與緩衝液 13 人類胚腎 (293T)細胞株培養 14 質體製備 14 勝任細胞製備 15 轉染作用 (Transformation) 15 轉移感染（Transfection） 15 點突變 16 聚合酶連鎖反應（Polymerasae Chain Reaction, PCR） 16 電生理紀錄-全細胞膜電箝紀錄(whole cell patch clamp) 16 資料分析 17 結果 18 鈣調素可增加Nav1.4電流密度 18 鈣調素N端有正常鈣離子結合能力時可增加Nav1.4電流密度 19 鈣離子濃度上升無助於Nav1.4單獨存在293T細胞時 20 鈣離子濃度上升有助於鈣調素增加Nav1.4電流密度 21 當鈣離子濃度上升有助於C-lobe失去鈣離子結合能力之鈣調素增加Nav1.4電流密度 22 當鈣離子濃度上升無助於失去鈣離子結合能力之鈣調素或只有N-lobe失去鈣離子結合能力之鈣調素增加Nav1.4電流密度 24 鈉離子通道IQ motif的各種突變 25 討論 27 鈣調素對Nav1.4的功效 27 鈣調素的鈣離子結合能力對Nav1.4的功效 28 鈣離子對鈣調素調控Nav1.4上的功效 29 IQ motif 突變會影響電壓依賴行鈉離子通道的活性 30 參考文獻 32 表 42 圖解 48 圖 506087732 bytesapplication/pdf論文公開時間：2015/07/29論文使用權限：同意有償授權(權利金給回饋本人)電壓依賴型鈉離子通道鈣調素鈣離子IQ motif全細胞膜片箝技術thesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/261678/1/ntu-103-R01B41008-1.pdf