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Title: | Shaker鉀離子通道S4區段之位移及其主要電阻區之位置 Molecular Movement of S4 and the Related Impedence Zone in Shaker Potassium Channels |
Authors: | Chia-Wei Chang 張佳偉 |
Advisor: | 郭鐘金 |
Keyword: | 鉀離子通道,主要電阻區,電位感測器, potassium channel,impedence zone,voltage sensor, |
Publication Year : | 2005 |
Degree: | 碩士 |
Abstract: | Shaker 鉀離子通道是一種電位開關性的離子通道,由四個相同的多肽鏈次單位所組成,其 C 端和 N 端都位在細胞膜內;每個次單位包含六條穿膜蛋白 (S1~S6),其中S5-S6 形成離子通道的孔洞;在 S4 上因為被發現有規則排列的鹼性胺基酸,因此被認為其和感測電場的變化有關,視為電位感測器。近年對於 S4 運動方式主要有兩種說法:第一種是 S4 利用旋轉並往細胞外位移的方式運動;第二種是 S4 位於通道蛋白質的外圍與細胞膜接觸,利用擺動的方式移動。本論文藉由 S4 上其他非鹼性胺基酸的突變,來探討包圍在S4外側之主要電阻區(即S4運動所必須行經以提供電能變化來開關通道者)之形狀與位置,並期能夠因而更加了解S4去極化時移動的方式與功能作用。在將 S4 上非鹼性胺基酸的位置一一突變成帶正電胺基酸 arginine;我們發現 I364R、L366R突變造成通道的活化速率與不活化速率很明顯變慢,在I364R突變觀察到速率之電壓依賴性顯著增強,L366R突變則否。將 364 和 366 兩個位置再做其他不同性質的突變(K、D、E、A、Q、W),發現I364位置突變成鹼性胺基酸時對電壓依賴性大致都有明顯增加。而L366位置的鹼性胺基酸突變對活化反應之電壓依賴性的影響都不大,但其不活化反應的電壓依賴性則皆變得極不顯著,而且最終不活化通道之比例皆大幅降低。至於I364及L366這兩個位置的若干突變,則都明顯有活化與去活化速率異常變慢的情形,似乎是大幅提高通道活化反應的能量峰值。從 V367一直到 L375 的非鹼性胺基酸位置突變成arginine記錄不到任何電流,到了S376R 與 H378R突變則又可記錄到電流,且其活化速率之絕對值與電壓依賴性與 WT 相較變化並不大。由 I364R、L366R 突變的結果,推測這兩個位置位在 S4 移動的過程會經過主要電阻區,而I364位置可以使鹼性胺基酸攜帶電荷,L366位置則否,由此可以推測他們所處的環境。綜合上述結果,我們描繪出箝住S4的主要電阻區其位置與形狀:在S4之3條”軸線”,即“R”、“R-1”、“R+1”線上,應分別經過R362(R軸線上)﹔L358、L361及/或I364(“R-1”軸線上)﹔V363與L366之間(“R+1”軸線上)。我們的數據同時也符合鉀離子通道在去極化過程中,其S4 可能移動 9 個胺基酸的距離,並轉動了180 度之想法。 Shaker K+ channel is a member of the voltage-gated K+ channels which are composed of four polypeptide subunits. Each of the subunits has six transmembrane segments (S1-S6), with the C- and N-termini both located intracellularly. Because of the regularly spaced basic amino acids, S4 has been considered as the voltage sensor of the channel. S4 presumably can move in response to membrane electric field change and thus cause subsequent gating conformational changes of the channel. There are chiefly two models explaining the S4 movement: the helical model and the paddle model. We made point mutation on S4 unchanged residues in S4, from V363 (which is located right internal to the outermost positive residue R362) to H378. In I364R and L366R mutant channels, the activation and inactivation rates are evidently slower. I364R shows more voltage-dependent gating parameters as compared to the wild type channels, but L366R did not. I364K has similar characteristics to I364R. The other mutations involving I364 (I364D, E, A, Q, W) do not show increased voltage dependence in gating parameters, although the activation and inactivation curve are shifted to different extent in the voltage axis. L366R and L366K not only show no increased voltage dependence in the gating parameters, but also has very slow activation kinetics. Markedly decreased voltage dependence in the inactivation kinetics. Some of the other mutations at L366 (L366D, E, W) also showed slowed gating kinetics. We could not detect any currents in single mutations involving the hydrophobic residues in S4 from V367R to L375R, but S376R and H378R give definite currents. We propose that I364 and L366 move across a significant part of electric field (impedence zone), whereas S376 probably does not cross the field during the gating movement of S4. The impedence zone goes through R362 (in the R axis), L358, L361 and/or I364 (in the “R-1” axis), and between V363 and L366 (in the “R+1” axis). Moreover, our data are also consistent with the proposal that S4 most likely moves through the gating canal by a translation shift of 9 residues and a rotation of ~180°during channel activation. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/35949 |
Fulltext Rights: | 有償授權 |
Appears in Collections: | 生理學科所 |
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