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研究生:陳映芝
研究生(外文):Ying-Chih Chen
論文名稱:Shaker鉀離子通道S4穿膜區段活化位置對應電荷之尋找
論文名稱(外文):The Counter Charges Coordinating with Arginines in S4 in Shaker Potassium Channel
指導教授:郭鐘金郭鐘金引用關係
指導教授(外文):Chung-Chin Kuo
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:生理學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2008
畢業學年度:96
語文別:中文
論文頁數:57
中文關鍵詞:Shaker鉀離子通道S4穿膜區段活化S3-S4連接區段對應電荷
外文關鍵詞:Shaker K+ channelS4activationS3-S4 linkercounter charge
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Shaker鉀離子通道是一種電位開關性離子通道,由四個次單位組成,每個次單位中含有六條穿膜蛋白 (S1-S6) 。離子通道的孔洞部分由S5-S6所構成,而S4上因為帶有一些規則排列的鹼性胺基酸,被認為可能與感測電場變化有關。當細胞膜去極化時,S4能感測電場變化而移動,進而使得通道孔洞開啟。目前關於S4的運動方式主要有兩種假設:一、S4以旋轉並往細胞外位移的方式移動。二、S4位於通道蛋白質的外圍與細胞膜接觸,並利用擺動的方式做運動。依第一種模式,當通道活化時,S4上的帶正電鹼性胺基酸可能會隨著S4的上移而移動至原本S3-S4 linker的位置。意味著在S3-S4 linker的某些位置上可能具有能穩定帶正電鹼性胺基酸的環境。由本實驗室之前的結果發現,將M356與A359突變成arginines之後,突變通道之活化、不活化曲線相對於正常通道都有明顯右移但斜率變化不顯著之情形。且其活化、不活化速率也較正常通道來得慢。推測這兩個位置極有可能含有能夠穩定arginines的極性或帶相反電荷的胺基酸存在。這樣的結果暗示著S4移動的過程當中除了休息態與活化態兩種狀態的位置外,可能還經過了兩個停靠站。因此在本實驗中,試圖在M356和A359位置附近找出可能穩定arginines的胺基酸。S2上的E283、E293,S5上的E418,以及S5-S6 loop上的E422、D431、D447。這些是位於S4之可能環境且演化上具高度保留性的極性或帶負電荷之胺基酸,因此應值得予以檢視其是否可能提供M356和A359一個穩定arginines的環境。我們利用site-directed mutagenesis將胺基酸突變,使之不具有極性或帶電性,並觀察其對M356R、A359R及M356RA359R突變離子通道的影響。而由目前的數據顯示,E283A改變了Shaker鉀離子通道活化的性質,且E283AM356R及E283AA359R對於E283A活化曲線及不活化曲線的偏移程度似乎比在野生型Shaker離子通道中來得小。暗示著E283A可能是在356、359位置周圍提供負電荷的酸性胺基酸之一。E422A與D431A則輕微減少M356R與A359R之偏移程度,但本身對鉀離子通道沒有顯著影響。而E293A突變幾乎不影響M356R與A359R活化曲線及不活化曲線之偏移。其他位置如E418及D447的極性或帶負電荷之胺基酸突變,由於未能表現電流,故其是否提供S4穿膜區段活化位置之對應電荷,尚待將來以其他方式繼續探討。
Shaker K+ channel is a member of the S4 voltage-gated ion channel superfamily which is composed of four polypeptide subunits. Each of the subunits contains six transmembrane segments (S1-S6). S5-S6 segments form the central pore, while S4 is considered as the voltage sensor of the channel and contains regularly spaced basic amino acids. Membrane depolarization causes S4 to undergo conformational changes that lead to opening of the channel pore. Currently, there are two different models of S4 movement: one is the helical screw model, and the other is the paddle model. According to the first model, manipulation of the S3-S4 linker may change the gating behavior because the S3-S4 linker is directly connected to S4. In fact, the Vh of the activation curves are shifted rightwardly when compared to the wild type channels in two point mutations in the S3-S4 linker, namely M356R and A359R. The rightwardly shifted activation curves and slower activation/inactivation rates suggest that some polar or acidic residues exist in the gating canal and interact with the basic side chains of the amino acids at positions 356 and 359. Possible candidates for these acidic residues are E283, E293 in S2; D316 in S3, E418 in S5; E422、D431、D447 in the S5-S6 loop. We use site-directed mutagenesis to neutralize these residues, and observe their effects on M356R, A359R, or M356RA359R mutants. We found that, mutation of E283 to alanine itself changes the gating behavior of the Shaker K+ channel. However the activation and inactivation curves of M356R and/or A359R shift less in the presence than in the absence of coexisting E283A mutation. We therefore consider E283 a possible acidic residue that provides a counter charge interacting with the basic side chains of arginines in S4. In contrast, there are negligible effects on E293 mutation on M356R, A359R, and M356RA359R mutant channels. E293 is thus unlikely one of the counter charges interacting with the basic residues in S4. Finally, replacement of E422 or D431 with alanine partially reduces the extent of the activation curve shift by M356R and A359R mutations. Therefore E422 and D431 may also play a role in the process of S4 movement.
誌謝 ii
中文摘要 iii
英文摘要 v
第一章 緒論 1
第二章 材料與方法 12
第三章 結果 16
第四章 討論 22

圖目次
圖1 Shaker 鉀離子通道之topology模式圖與胺基酸序列 31

圖2 WT IR、M356R IR、A359R IR及M356RA359R IR之活化電流紀錄與活化曲線 32

圖3 WT IR、E283A IR、E283AM356R IR、E283AA359R IR及E283AM356RA359R IR之活化電流紀錄與活化曲線 33

圖4 WT IR、E293A IR、E293AM356R IR、E293AA359R IR及E293AM356RA359R IR之活化電流紀錄與活化曲線 34

圖5 WT IR、E422A IR、M356RE422A IR、A359RE422A IR及M356RA359RE422A IR之活化電流紀錄與活化曲線 35

圖6 WT IR、D431A IR、M356RD431 IR、A359RD431 IR及M356RA359RD431 IR之活化電流紀錄與活化曲線 36

圖7 WT、M356R、A359R及M356RA359R之不活化電流紀錄與不活化曲線 37

圖8 WT、E283A、E283AM356R、及E283AA359R之不活化電流紀錄與不活化曲線 38

圖9 WT、E293A、E293AM356R、E293AA359R及E293AM356RA359R之不活化電流紀錄與不活化曲線 39

圖10 WT、E422A、M356RE422A、A359RE422A及M356RA359RE422A之不活化電流紀錄與不活化曲線 40

圖11 WT、D431A、M356RD431A、A359RD431A及M356RA359RD431A之不活化電流紀錄與不活化曲線 41

表目次
表1 各突變通道活化曲線之Vh值及k值 42
表2 各突變通道不活化曲線之Vh值及k值 43

參考文獻 44
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