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標題: | 電壓感應開啟鉀離子通道與蜘蛛毒蛋白間的動力學研究 Kinetic study of interactions between voltage-gated K+-channels and toxins from spider venom |
作者: | Yu-Wen Shih 石又文 |
指導教授: | 樓國隆 |
關鍵字: | 電壓感應開啟式鉀離子通道,停止流螢光光譜分析儀,電壓感受體, Voltage-gated potassium channel,Stopped-flow spectrofluorimeter,voltage sensor, |
出版年 : | 2005 |
學位: | 碩士 |
摘要: | 電壓感應開啟式鉀離子通道(Voltage-gated potassium channel, Kv channel)普遍存在於各類組織,主要負責在細胞膜興奮時開啟,造成再極化。通常由四個次單元體組成,每個次單元體上含有通道形成區域(pore-forming domain)及四個電壓感受過膜片段(voltage-sensing transmembrane segments),稱之為S1-S4。其中S4是最主要的電壓感受體(voltage sensor)。又目前已知S3的C端(S3C)可與通道開啟調整毒素,如蜘蛛毒蛋白(Hanatoxin1, HaTx1;Stromatoxin1, ScTx1)作用使通道開啟時所需電壓改變。而最常被用來研究S3C 結構-功能之電壓感應開啟式鉀離子通道Kv 2.1 channel(drk1)屬shab family,會受蜘蛛毒蛋白作用而不易開啟;相對地,shaker(Kv1.1 channel)則不受蜘蛛毒蛋白之影響。S3C 可能之二級結構也因此透過相關研究而有逐步瞭解,且目前一般相信S3C含有一段獨立的α-helix。
經由本實驗室前人對電壓感應開啟式鉀離子通道S3C與蜘蛛毒蛋白所做三度空間結構上的分析,及利用分子模擬與嵌合的技術,可得到電壓感應開啟式鉀離子通道S3C與蜘蛛毒蛋白交互作用之分子模型。近幾年,由Roderick Mackinnon所帶領的研究團隊對於電壓感應開啟式鉀離子通道之電壓感受體的研究有一些異於傳統之突破。他們利用古溫泉細菌Aeropyrum pernix之電壓感應開啟式鉀離子通道(KvAP)的蛋白質單晶,以X光繞射得到結構及配合一系列與電壓感受體相關的電生理實驗研究,提出不同於傳統Kv channel結構之模型,並且利用序列同源性比對,將此一模型推廣適用到大部分真核生物之電壓感應開啟式鉀離子通道上。此模型與之前的許多真核生物之電壓感應開啟式鉀離子通道的電生理實驗結果未盡相符,亦與本實驗室前人所做之作用機制模型的基礎假設有所差異。 因此,在本論文中,筆者以之前本實驗室前人所完成的關於Kv2.1 S3C與Hanatoxin結合時之模擬計算為基礎,透過停止流螢光光譜(stopped-flow),對S3C片段與Hanatoxin的作用,進行了一系列的實驗分析與探討。所得到的生化動力學數據,可以作為檢驗先前提出的分子機制及探討S3C片段之生理可能構形時之重要依據。配合上先前之模擬計算,我們也對此一作用發生時各分子在細胞膜上空間分佈的關係作了深入的推理。 透過Hanatoxin或Stromatoxin與Kv2.1 S3C片段結合的動力學實驗,可以計算出結合發生時之kon、koff值。比較了使用Kv1.1和Kv2.1 S3C 變異株作為控制組的結果後,若再配合加入TFE(2,2,2-trifluoroethanol)使Kv2.1 S3C片段重新摺疊(refolding),可以清楚得知, Kv2.1 S3C片段與Hanatoxin之結合可能確實依照先前分子模擬預測之機制進行。至於Kv2.1 S3C不同變異株(hydrophobic variant/hydrophilic variant)之間的比較部分,我們則得到稍異於分子模擬預測之結果:疏水性與親水性的作用應同等重要,突變任一類鍵結所需殘基均能使蜘蛛毒蛋白與Kv2.1 S3C 片段之結合能力消失,而非如之前預測的由極性反應擔負較重要之角色。綜合以上生化動力學實驗並配合分子模擬預測之結果,加上先前諸多電生理的資料,我們推測負責與Hanatoxin鍵結之Kv2.1 S3C片段殘基應位於細胞膜邊界處,靠近磷脂(phospholipids)之hydrophilic head處。因此整個Kv2.1 S3C 片段必須以稍稍傾斜的方式坐落於細胞膜之外緣。因此傳統的結構模型,由於指出了external crevice的特性,似乎較符合此處的整體空間關係。至於Stromatoxin,我們認為在序列上與Hanatoxin十分接近,並且同為抑制Kv2.1 channel之毒蛋白,故應可得到類似於Hanatoxin之生化動力學實驗結果;但實際上所得之數據卻傾向以迥異於對Hanatoxin之分析來詮釋。由於Stromatoxin是一種近年來新發現之蜘蛛毒蛋白,功能測試之數據並不完整,僅知其對於Kv2.1擁有如Hanatoxin一般使通道趨於不易開啟之類似特性,其抑制Kv2.1 channel之詳細作用機制則仍不十分明瞭,但相信其細部摺疊在生理特性上應有重要意義。這些都有待更深入地研究以釐清其中的關鍵因素。 本論文不僅將先前關於蜘蛛毒與Kv通道反應機制之研究以生化實驗給予相當程度的驗證,也呈現了一些新且有趣的現象,有待更進一步的觀察與討論。 Voltage-gated potassium channels are found in a wide variety of tissues, where their primary role is to respond to the membrane excitation to allow the repolarization phase of an action potential to occur and therefore the K+ ions can efflux. Such channels are normally homotetramers and each subunit contains four voltage-sensing transmembrane segments, namely S1 through S4, whereas S5 and S6 form the pore. Among them, S4 may play the most crucial role in sensing the voltage change. Kv2.1, a member of shab potassium channel family, is one the most commonly applied channels in study of the structural-functional correlation for voltage sensing. Upon binding of hanatoxin, the midpoint of the curve for required gating potential of Kv2.1 can be shifted to the right, which means more difficult to open the channels under the same condition. On the contrary, shaker potassium channels do not show such effect. The secondary structural arrangement of S3C has been, due to such studies, intensively analyzed and the existence of an independent α-helix was then suggested. It has been accomplished to establish a model and hypothesis describing the molecular details of hanatoxin-binding induced gating. This was based on the 3-D structural analysis with molecular docking and simulations. Recently, research by Mackinnon’s group has led to certain controversial developments in the voltage-sensing theory of Kv channels. Crystal structure of KvAP channel from archaeum Aeropyrum pernix revealed, combined with a series of electrophysiological experiments and sequence comparisons, a novel ‘voltage-sensor paddle’ model, which was anticipated to be applied on eukaryotic Kv channels. However, such revolutionary idea did challenge the conventional “translocation” model and contradict to a few previously acknowledged experimental results. Such discovery has also brought gross impact on our proposed mechanism which was based upon the conventional translocation model in Kv channels. Therefore, in this thesis, we have performed the kinetic analysis with stopped-flow to examine our previously proposed hypothesis. Such binding study, in combination with related calculations, provides further possibility to consider in a more decent way the discussion of the reasonable conformation and membrane distribution of S3C segment in the toxin-Kv channel interactions. The binding rate constant kon and release rate constant koff for interactions between hanatoxin/stromatoxin and Kv2.1 S3C segments can be calculated through the kinetic analysis with stopped-flow. Upon utilization of Kv2.1 S3C mutants and Kv1.1 S3C as control experiments, together with the appropriate additions of trifluoroethanol in different concentrations, which allow the refolding of Kv2.1 S3C to occur, it has been indicated that binding of hanatoxin and Kv2.1 S3C may follow the molecular details described in our proposed mechanism. However, the comparison between the hydrophobic and hydrophilic interactions required for binding between hanatoxin and Kv2.1 S3C observed from rationally designed mutants (hydrophobic part v.s. polar part of residues) suggests that both types of interaction are equally crucial for binding. Mutation of either part of residues can result in the abolishment of binding ability for hanatoxin and Kv2.1 S3C. Polar interactions should not be the only dominant factor able to affect such binding as predicted thru simulation study. All together, it is reasonable to comprehend that the S3C residues required for binding with hanatoxin should be located at the boundary of cell membrane, nearby the hydrophilic heads of phospholipids (or interfacial area of external face) with a slight tilting angle. Therefore the conventional ‘translocation’ model may fulfill such requirement better, especially considering the spatial orientations of transmembrane segments around the external crevice. On the other hand, due to the sequence and functional similarity between stromatoxin and hanatoxin, we did expect that the kinetic data for stromatoxin present themselves in a very similar pattern as those of hanatoxin do. However, surprisingly, all the observations tend to draw our attention a totally different way of interpretation. Probably the structural details, rather than general structural feature, of stromatoxin may play pivotal role in the binding behavior. This awaits further investigations. This thesis provides experiments support on our previously proposed molecular mechanism of tarantula toxins binding induced gating in Kv channels to a certain extent. It also leads to some interesting questions open for future study. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/35300 |
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顯示於系所單位: | 口腔生物科學研究所 |
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