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  1. NTU Theses and Dissertations Repository
  2. 生命科學院
  3. 生命科學系
請用此 Handle URI 來引用此文件: http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/57686
標題: 鈣調素對電壓依賴型鈉離子通道Nav1.4的影響
The Effects of Calmodulin on the Voltage-gated Sodium Channels, Nav1.4
作者: Yi-Ting Tsai
蔡依廷
指導教授: 潘建源(Chien-Yuan Pan)
關鍵字: 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,
Nav1.4,CaM,Ca+2,IQ motif,whole cell patch clamp,
出版年 : 2014
學位: 碩士
摘要: 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.
URI: http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/57686
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