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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 湯志永(Chih-Yung Tang) | |
| dc.contributor.author | Jui-Hsiang Hu | en |
| dc.contributor.author | 胡瑞香 | zh_TW |
| dc.date.accessioned | 2021-06-15T06:59:40Z | - |
| dc.date.available | 2016-03-03 | |
| dc.date.copyright | 2011-03-03 | |
| dc.date.issued | 2010 | |
| dc.date.submitted | 2011-01-25 | |
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Nature 420, 193-198. Ziechner U, Schonherr R, Born AK, Gavrilova-Ruch O, Glaser RW, Malesevic M, Kullertz G & Heinemann SH. (2006). Inhibition of human ether a go-go potassium channels by Ca2+/calmodulin binding to the cytosolic N- and C-termini. FEBS J 273, 1074-1086. Zimmermann S, Hartje S, Ehrhardt T, Plesch G & Mueller-Roeber B. (2001). The K+ channel SKT1 is co-expressed with KST1 in potato guard cells--both channels can co-assemble via their conserved KT domains. Plant J 28, 517-527. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/48506 | - |
| dc.description.abstract | 大鼠的ether-à-gogo ( rEag ) 電位控制 ( voltage-gated ) 鉀離子通道屬於EAG家族的一種,可細分為rEag1與rEag2兩個類型。rEag1和rEag2是只表達於神經系統 ( neuron-specific ) 的鉀離子通道,所以對調控神經細胞的興奮性可能扮演重要角色。
一個具有功能性的電位控制鉀離子通道是由四個次單元( subunit )所聚合而成的四聚體 ( tetramer )。過去的研究顯示鉀離子通道之次單元之間的聚合可由特定的胺基酸序列調控來決定彼此之間的專一性或特異性,進而形成四聚體。例如在Kv1鉀離子通道目前研究已知其主要聚合區( assembly domain )位於細胞內N端 ( amino terminus ) 之T1區( tetramerization domain )。對rEag1鉀離子通道而言,目前的研究指出在其C端 ( carboxyl terminus ) 有一段包含41個胺基酸 ( 胺基酸897-937 ) 的區域,對rEag 鉀離子通道的次單元之間之聚合作用扮演重要角色,所以此一C端此區被稱為C端聚合區域( C-terminal assembly domain;CAD )。然而近年有報告顯示,對另一種EAG家族的鉀離子通道Herg而言,N端的部分序列可能與次單元的聚合機制有關。所以本篇論文的研究目的是要探討rEag1的CAD片段是否的確是決定其聚合作用的關鍵部位。 我們以rEag1正常型 ( wild-type ) 當模版,利用分子生物 mutagenesis方法產生十九種長短不同的刪除突變 ( truncation mutants ) ,並經由雙極電位箝制術 ( two-electrode voltage clamp ) 記錄判定各種不同的突變次單元是否能單獨聚合為具功能性的鉀離子通道。對於不具有功能性的突變種鉀離子通道,我們則將其以共同表現的方法 ( co-expression ) ,決定是否能對正常型rEag1次單元造成顯性抑制作用 ( Dominant-negative effect ) 。 根據我們的實驗結果證實兩個完全不具有CAD的刪除突變,確實能自行產生與正常型rEag1相似的鉀離子通道。另外,即使將缺乏C端刪除了485個胺基酸的突變也可以對正常型rEag1鉀離子通道產生顯著抑制作用。這樣的結果我們可以推論CAD對rEag1鉀離子通道的聚合機制來說,可能不是扮演著最主要或必要的角色;另外,除了S6以外,N端與大部份的穿膜段落也可能不是負責rEag1鉀離子通道聚合機制的主要區域。所以我們的實驗結果可提供未來研究rEag1鉀離子通道聚合機制的一個新方向。 | zh_TW |
| dc.description.abstract | The voltage-gated rat ether-à-gogo (rEag) potassium channel is a member of the EAG potassium channel family, and can be subdivided into two different subtypes: rEag1 and rEag2. rEag1 and rEag2 potassium channels have been shown to express exclusively in neurons, and are therefore thought to play an important role in determining the membrane excitability of neurons.
A functional voltage-gated potassium channel is a tetramer comprising the assembly of four subunits. The assembly of potassium channel subunits is usually mediated by specific amino acid sequence that dictates the specificity of tetramer formation within each potassium channel family. For example, the assembly of Kv1 channel is primarily determined by a region within the intracellular amino terminus (N-terminus) known as the tetramerization domain (T1-domain). For rEag1 potassium channel, a 41-amino-acid domain (amino acids 897-937), close to the carboxyl terminus (C-terminus), has been shown to be important for rEag1 potassium channel subunit interaction. Therefore, this C-terminal domain was named the C-terminal assembly domain (CAD). However, recent reports have demonstrated that in Herg channels, another member of the EAG potassium channel family, the N-terminus may contribute to the specific interaction between different splice variants. Therefore, in this study we aim to investigate whether the CAD of rEag1 is indeed the key assembly domain. More than 10 different truncation mutants, all of which without the CAD, have been introduced into rEag1 potassium channels. By using the two-electrode voltage clamp technique, we first determined whether some of these truncation mutants produced functional potassium channels. For nonfunctional mutants, we then decided if they displayed dominant-negative effects on wild-type rEag1 channels by performing co-expression experiments. Our electrophsyiological studies demonstrated that two truncation mutants lacking the CAD indeed generated functional potassium channels. In addition, significant dominant-negative effects on WT-rEag1 channels were observed for non-functional mutants with truncations up to 485 amino acids in the C-terminal region. These data indicate that the role of the CAD needs to be re-defined and that the S6 segment and the proximal region of C-terminus may contribute to the assembly of rEag1 channels. Our new findings may pave the way for a new direction for understanding the assmbly mechanism of EAG channel family. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-15T06:59:40Z (GMT). No. of bitstreams: 1 ntu-99-R95441008-1.pdf: 1531564 bytes, checksum: b10982520766373ce5edc232588fa0b9 (MD5) Previous issue date: 2010 | en |
| dc.description.tableofcontents | 誌謝 ‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥I
目錄 ‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥III 圖次 ‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥V 表次 ‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥VII 中文摘要 ‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥VIII 英文摘要 ‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥X 第一章、 導論‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥1 I. 鉀離子通道概論‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥1 II. Eag鉀離子通道介紹‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥2 II-1. 種類 ‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥2 II-2. 結構 ‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥3 II-3. Gating mechanism ‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥4 III. Assembly of K+ channel subunits‥‥‥‥‥‥‥‥‥‥4 III-1. Kv鉀離子通道的聚合(assembly)作用 ‥‥‥‥‥‥‥4 III-2 其他離子通道之聚合機制‥‥‥‥‥‥‥‥‥‥‥‥‥‥8 III-3 輔助性蛋白之聚合作用‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥13 III-4. EAG鉀離子通道的聚合作用 ‥‥‥‥‥‥‥‥‥‥‥‥14 IV. 本實驗之研究目的 ‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥16 第二章、 材料與方法 ‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥18 I. Molecular Biology‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥18 I-1. cDNA clones ‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥18 I-2. Construction of truncation mutants ‥‥‥‥‥‥‥18 I-3. Transformation and plasmid DNA extraction ‥‥‥‥23 I-4. In Vitro Transcription‥‥‥‥‥‥‥‥‥‥‥ ‥‥23 II. 電生理紀錄‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥ ‥‥25 II-1. Xenopus laevis ( 南非爪蟾 ) oocyte之取得與處理‥‥26 II-2. cRNA injection‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥27 II-3. Co-injection of different cRNA constructs‥‥‥‥28 II-4. 電生理紀錄‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥28 II-5. 電生理紀錄之分析‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥29 第三章、 結果‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥31 I. 檢視rEag1、Myc-rEag1和rEag1-HA channel之生物物理特性31 II. 缺乏CAD 時仍可能形成具功能性的rEag1鉀離子通道‥‥‥33 III. 缺乏CAD 之C-terminus truncation mutants仍可能可與WT rEag1 channel形成co-assembly‥‥‥‥‥‥‥‥‥‥‥‥‥‥34 IV. N端與大部分的transmembranes domain可能不是負責媒介rEag1 channel assembly的主要區‥‥‥‥‥‥‥‥‥‥‥‥‥36 第四章、 討論‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥38 I. 探討rEag1可能的assembly domain位置‥‥‥‥‥‥‥‥‥38 II. CAD 在rEag1 channel assembly之角色‥‥‥‥‥‥‥‥‥40 III. 後續進一步的實驗方向‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥44 結論‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥45 參考文獻‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥79 | |
| dc.language.iso | zh-TW | |
| dc.subject | 顯性抑制作用 | zh_TW |
| dc.subject | 雙極電位箝制術 | zh_TW |
| dc.subject | 聚合區 | zh_TW |
| dc.subject | 聚合機制 | zh_TW |
| dc.subject | 次單元 | zh_TW |
| dc.subject | 四聚體 | zh_TW |
| dc.subject | EAG家族 | zh_TW |
| dc.subject | 電位控制 | zh_TW |
| dc.subject | 大鼠Eag1鉀離子通道 | zh_TW |
| dc.subject | assembly mechanism | en |
| dc.subject | CAD | en |
| dc.subject | C-terminal assembly domain | en |
| dc.subject | Dominant-negative effect | en |
| dc.subject | two-electrode voltage clamp | en |
| dc.subject | assembly domain | en |
| dc.subject | -gogo | en |
| dc.subject | assembly | en |
| dc.subject | tetramer | en |
| dc.subject | subunit | en |
| dc.subject | rEag1 | en |
| dc.subject | rat | en |
| dc.subject | ether-&agrave | en |
| dc.title | 大鼠Eag1鉀離子通道之聚合機制 | zh_TW |
| dc.title | Mechanistic examination of the assembly of rat Eag1 potassium channel | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 99-1 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 郭鐘金(Chung-Chin Kuo),符文美(Wen-Mei Fu) | |
| dc.subject.keyword | 大鼠Eag1鉀離子通道,電位控制,EAG家族,四聚體,次單元,聚合區,聚合機制,雙極電位箝制術,顯性抑制作用, | zh_TW |
| dc.subject.keyword | rEag1,rat,ether-&agrave,-gogo,assembly,assembly mechanism,tetramer,subunit,assembly domain,two-electrode voltage clamp,Dominant-negative effect,C-terminal assembly domain,CAD, | en |
| dc.relation.page | 103 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2011-01-25 | |
| 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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