DC 欄位 | 值 | 語言 |
dc.contributor | 指導教授:潘建源 | - |
dc.contributor | 臺灣大學:生命科學系 | zh_TW |
dc.contributor.author | 蔡依廷 | zh_TW |
dc.contributor.author | Tsai, Yi-Ting | en |
dc.creator | 蔡依廷 | zh_TW |
dc.creator | Tsai, Yi-Ting | en |
dc.date | 2014 | - |
dc.date.accessioned | 2014-11-26T02:15:43Z | - |
dc.date.accessioned | 2018-07-06T01:22:34Z | - |
dc.date.available | 2014-11-26T02:15:43Z | - |
dc.date.available | 2018-07-06T01:22:34Z | - |
dc.date.issued | 2014 | - |
dc.identifier.uri | http://ntur.lib.ntu.edu.tw//handle/246246/261678 | - |
dc.description.abstract | 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。 | zh_TW |
dc.description.abstract | 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. | en |
dc.description.tableofcontents | 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 50 | zh_TW |
dc.format.extent | 6087732 bytes | - |
dc.format.mimetype | application/pdf | - |
dc.language | zh-TW | - |
dc.rights | 論文公開時間:2015/07/29 | - |
dc.rights | 論文使用權限:同意有償授權(權利金給回饋本人) | - |
dc.subject | 電壓依賴型鈉離子通道 | zh_TW |
dc.subject | 鈣調素 | zh_TW |
dc.subject | 鈣離子 | zh_TW |
dc.subject | IQ motif | zh_TW |
dc.subject | 全細胞膜片箝技術 | zh_TW |
dc.title | 鈣調素對電壓依賴型鈉離子通道Nav1.4的影響 | zh_TW |
dc.title | The Effects of Calmodulin on the Voltage-gated Sodium Channels, Nav1.4 | en |
dc.type | thesis | en |
dc.identifier.uri.fulltext | http://ntur.lib.ntu.edu.tw/bitstream/246246/261678/1/ntu-103-R01B41008-1.pdf | - |
item.openairecristype | http://purl.org/coar/resource_type/c_46ec | - |
item.openairetype | thesis | - |
item.grantfulltext | open | - |
item.cerifentitytype | Publications | - |
item.fulltext | with fulltext | - |
顯示於: | 生命科學系
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