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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 湯志永 | |
dc.contributor.author | Ssu-Ju Fu | en |
dc.contributor.author | 傅斯如 | zh_TW |
dc.date.accessioned | 2021-06-17T00:53:33Z | - |
dc.date.available | 2017-03-02 | |
dc.date.copyright | 2012-03-02 | |
dc.date.issued | 2011 | |
dc.date.submitted | 2011-10-17 | |
dc.identifier.citation | Akhavan A, Atanasiu R & Shrier A. (2003). Identification of a COOH-terminal segment involved in maturation and stability of human ether-a-go-go-related gene potassium channels. J Biol Chem 278, 40105-40112.
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/66722 | - |
dc.description.abstract | EAG(ether-á-go-go)鉀離子通道屬於電性控制(voltage-gated)型鉀離子通道。而EAG鉀離子通道家族可分為三個不同次家族,包括Eag、Erg,以及ElK。其中Eag次家族又可再細分為Eag1與Eag2兩種類型。
一個有功能的電位控制鉀離子通道是由四個α次單元所組成的四聚體,而且鉀離子通道α次單元間的聚合,必須經由次單元上特定的蛋白質區域所媒介。對rat Eag1(rEag1)鉀離子通道來說, C端(carboxyl terminal)的carboxyl assembly domain(CAD)常被推測是與rEag1離子通道的聚合作用有關。但是本實驗室先前的研究發現,兩個不具有CAD卻仍具有鉀離子通道功能的刪除突變。而且我們也發現數個缺少CAD但保有S6 segment和proximal carboxyl region不具有功能性的rEag1刪除突變,會對正常型rEag1 channel(rEag1-WT)產生顯著的抑制作用。因此,我們推測CAD並非rEag1離子通道主要的聚合結構。本篇論文的目的就是要延續前述的實驗結果,進一步討論EAG K+channel family 之聚合機制。 而本篇研究中,我們在rEag1和rEag2 channel又各發現了兩個不具有CAD但具有離子通道功能的刪除突變,再次驗證CAD非為rEag1重要的聚合區域。 此外,我們經由研究一系列不具有功能的刪除突變對WT channel的顯著抑制作用,並推論:對rEag1與rEag2 channel而言,S6 distal segment和proximal carboxyl terminal可能是最主要的聚合區域。我們還提出進一步證據顯示rEag1和rEag2形成heterotetramer時,可能也是經由類似的機制構成subunit。 為了在Human Erg(Herg) K+ channel次家族中延伸探討我們所提出的聚合機制,所以在Herg channel S6以及C-linker設計了數個刪除突變。令人意外的,當Herg-WT與S6上的刪除突變共同表現時,並未有任何顯著抑制作用,反而是具有完整C-linker的刪除突變引起了顯著的抑制作用。我們的數據顯示C-linker參與了通道次單元的聚合作用有關,但S6 segment則似乎較不相關。 在先前的研究顯示,Eag與Herg次家族中的成員無法co-assemble形成heterotetramer。為了探究subfamily-specific assembly的機制,我們分別將rEag1與Herg的刪除突變與rEag1-WT及Herg-WT共同表現,觀察是否有抑制WT channel的情形。有趣的是,含有 C-linker的Herg刪除突變會對rEag1產生顯著性抑制作用;但是,我們手中所有的rEag1刪除突變都不會對Herg-WT產生作用。目前的實驗結果我們無法確定rEag1與Herg之間是否真的具有交互作用,我們還需進行更多的證據來判定rEag1與Herg channel是否有可能產生交互作用。 | zh_TW |
dc.description.abstract | The ether-á-go-go (EAG) potassium (K+) channel family belongs to the superfamily of the voltage-gated K+ channel. The EAG K+ channel family can be divided into three subfamilies: Eag, Erg, and Elk. In mammals, the Eag subfamily can be further divided into two subtypes: Eag1 and Eag2.
A functional voltage-gated K+ channel comprises four pore-forming α subunits, and the assembly of the four α subunits into a tetrameric complex requires the presence of specific recognition domain within the channel protein. For rat Eag1 (rEag1) K+ channels, a short carboxyl assembly domain (CAD) in the carboxyl terminus (C-terminus) was originally suggested to be the assembly domain of rEag1 channels. A recent study from our lab, however, identified two CAD-lacking rEag1 truncation mutants that formed functional K+ channels. Furthermore, the same report also demonstrated that several CAD-lacking nonfunctional truncation mutants exerted significant dominant-negative effects on the functional expression of rEag1 wild-type (rEag1-WT) channels. Together, these observations indicate that CAD may not be essential for the assembly of rEag1 subunits. In light of this new finding, the purpose of this master thesis is to test the hypothesis that the non-CAD assembly mechanism originally put forth for rEag1 channels may be applicable to other members of the EAG K+ channel family. Here we have found four new CAD-lacking truncation mutants (two in rEag1 and the other two in rEag2) that produced functional K+ channels in Xenopus oocytes, further supporting the idea that CAD is not the assembly domain of EAK K+ channel family. In addition, by studying the dominant-negative effects of a series of different non-functional truncation mutants, we have provided further evidence suggesting that the distal S6 segment and the proximal carboxyl terminus (C-linker region in particular) may contribute to the assembly of rEag1 as well as rEag2 channels. Interestingly, our data further implied that the same protein region may also be implicated in the formation of rEag1-rEag2 heterotetramers. We have also extended the scope of our investigation to the assembly mechanism of the human Erg (Herg) K+ channels by generating several Herg truncation mutants over the S6 segment and the C-linker region. Unexpectedly, our study revealed that none of the Herg S6 truncation mutants exerted significant dominant-negative effects on the functional expression of Herg-WT channels. By contrast, a truncation mutant harboring an intact C-linker region displayed a potent dominant-negative effect. These data suggest the C-linker region, but not the distal S6 segment, may contribute to the assembly of Herg K+ channels. It has been previously shown that members of the Eag and Herg subfamilies failed to co-assemble into heteroteramers. To address the mechanism of this subfamily-specific assembly, we tested whether rEag1 and Herg truncation mutants may suppress the functional expression of rEag1-WT and Herg-WT, respectively. Surprisingly, the foregoing C-linker-containing Herg truncation mutant was found to display dominant-negative effects on rEag1-WT. Nevertheless, none of the rEag1 truncation mutants tested affected the functional expression of Herg-WT. Future experiments will be required to assess the structural significance of these apparently paradoxical results. | en |
dc.description.provenance | Made available in DSpace on 2021-06-17T00:53:33Z (GMT). No. of bitstreams: 1 ntu-100-R98441015-1.pdf: 5213115 bytes, checksum: fb53932a1ce971f6904984f29f55c5f7 (MD5) Previous issue date: 2011 | en |
dc.description.tableofcontents | 第一章 導論------------------------------------------------------------------------------------1
Ⅰ-1. 鉀離子通道概論--------------------------------------------------------------------1 Ⅰ-2. Interduction of EAG+ channel-----------------------------------------------------2 Ⅰ-3. Structure of EAG K+ channel------------------------------------------------------3 Ⅰ-4. Physiology and pathophysiology of EAG K+ channel-------------------------4 Ⅰ-5. Biophysical properties of Eag K+ channel---------------------------------------5 Ⅰ-6. Physiology and pathophysiology of Erg K+ channel---------------------------7 Ⅰ-7. Biophysical properties of Erg K+ channel----------------------------------------8 Ⅰ-8. Assembly of voltage-gated potassium channel---------------------------------10 Ⅰ-9. Erg subunit assembly--------------------------------------------------------------12 Ⅰ-10. Eag subunit assembly---------------------------------------------------------------13 Ⅰ-11. 實驗研究目的----------------------------------------------------------------------14 第二章 材料與方法--------------------------------------------------------------------------15 Ⅱ-1. cDNA clones------------------------------------------------------------------------15 Ⅱ-2. Construction of truncation mutants----------------------------------------------15 Ⅱ-3. Transformation and plasmid DNA extraction----------------------------------20 Ⅱ-4. In vitro transcription---------------------------------------------------------------20 Ⅱ-5. Xenopus laevis (南非爪蟾) oocyte之取得------------------------------------23 Ⅱ-6. Xenopus laevis (南非爪蟾) oocyte之處理------------------------------------24 Ⅱ-7. cRNA injection---------------------------------------------------------------------24 Ⅱ-8. Co-injection of different cRNA constructs-------------------------------------25 Ⅱ-9. 電生理紀錄-------------------------------------------------------------------------26 Ⅱ-10. 電生理紀錄之分析----------------------------------------------------------------26 第三章 結果----------------------------------------------------------------------------------29 Ⅲ-1. 新發現之缺少CAD但具有離子通道功能的rEag1刪除突變-----------29 Ⅲ-2. rEag1之S6刪除突變I467X為能夠產生顯著抑制作用的最短結 構-------------------------------------------------------------------------------------31 Ⅲ-3. 兩個缺少C端CAD結構之rEag2刪除突變,亦能形成具有功能性的離 子通道------------------------------------------------------------------------------32 Ⅲ-4. rEag2之S6 segment刪除突變也能夠產生顯著抑制作用-----------------34 Ⅲ-5. rEag2之proximal carboxyl terminus刪除突變也能夠產生顯著抑制 作用---------------------------------------------------------------------------------35 Ⅲ-6. Herg channel 之生物物理特性--------------------------------------------------36 Ⅲ-7. Herg之S6刪除突變並無法產生顯著抑制作用,C-linker刪除突變則 可以---------------------------------------------------------------------------------38 Ⅲ-8. rEag1與rEag2可能也由S6 distal segment以及proximal carboxyl terminus產生交互作用---------------------------------------------------------39 Ⅲ-9. Herg與rEag1 channel之間是否可產生交互作用尚無法確定-----------40 第四章 討論----------------------------------------------------------------------------------41 Ⅳ-1. CAD在rEag以及Herg聚合作用時所扮演的角色------------------------41 Ⅳ-2. S6 distal segment和C-linker在rEag1及rEag2聚合作用所扮演的 角色---------------------------------------------------------------------------------41 Ⅳ-3. Herg聚合機制之探討-------------------------------------------------------------42 Ⅳ-4. 討論rEag1和Herg-WT間是否可co-assembly------------------------------43 Ⅳ-5. 帶進一步釐清的問題以及其後續的實驗設計-------------------------------43 第五章 結論--------------------------------------------------------------------------------47 參考文獻--------------------------------------------------------------------------------------93 | |
dc.language.iso | zh-TW | |
dc.title | EAG鉀離子通道家族之聚合機制 | zh_TW |
dc.title | The subunit assembly mechanism of the EAG K+ channel family | en |
dc.type | Thesis | |
dc.date.schoolyear | 100-1 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 郭鐘金,符文美 | |
dc.subject.keyword | EAG鉀離子通道家族, | zh_TW |
dc.subject.keyword | rEag1,rEag2,Herg,EAG K+ channel, | en |
dc.relation.page | 102 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2011-10-18 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 生理學研究所 | zh_TW |
顯示於系所單位: | 生理學科所 |
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