臺灣博碩士論文加值系統

Shaker鉀離子通道是一種電位依賴性的離子通道，具有四個相同的次單元。每個次單元為一多肽鏈，其N端和C端都位在細胞膜內側，並且具有六個穿膜區段S1~S6形成通道的孔洞部份，而S1~S4是感應電位變化的部份。在S4上面具有排列規則的鹼性胺基酸，所以被認為是電位感測器，能感應電位變化來控制鉀離子通道的開關。此種鉀離子通道開啟後會進入不活化態，不活化的機制可以分成兩種：P/C型和N型不活化態。P/C型不活化發生的速度較N型不活化慢，可能是由於孔洞外口區形變使離子無法通過；N型不活化的發生是由N端胺基酸形成的鏈球結構所造成的，它會接上孔洞的結合位置讓孔洞發生形變使離子無法通過。由之前的實驗知道，在L366W上不活化球的存在與否會對其活化曲線有很大的影響；又L366R與L366K的不活化速率的電位依賴性變的很弱，而不活化速率的電位依賴性來自於活化與不活化的偶合。所以在本文中將S4上的L366以及N端不活化球上的胺基酸做點突變或雙點突變，藉此來探討L366位置與N端不活化閘門的作用。在L7、Y8上點突變成帶電胺基酸後，發現其活化曲線與WT相似但快速不活化行為消失，而在A3、V4、A5、G6這幾個位置上面點突變成帶電胺基酸，其不活化行為與WT有顯著差異，包括有不活化曲線Vh值右移、k值變大或不活化速率緩慢並且殘餘電流與峰值電流的比例還是較大這些現象，而在G9、L10、G11這三個位置上點突變成帶電胺基酸後，對於不活化行為的影響較弱。L366D單突變後，其活化曲線Vh值比起WT向左移動約37mV，但是k值並沒有顯著差異，可是其不活化曲線的k值卻增加到約為WT的兩倍。我們發現在N端不活化球或是L366上作單突變，其活化與不活化的偶合程度變差了；但是在N端不活化球上3-8位置突變成R且與L366D做雙突變後，由以下這些方面發現這些雙突變似乎使得活化與不活化的偶合程度比起單突變較好且與WT類似，包括有：原始電流紀錄圖上電流峰值下降並且出現交叉的情形、不活化曲線的k值及電流峰值與殘餘電流的比值與WT類似。由實驗中的發現，我們認為N端不活化球與L366這個位置具有交互作用，並且對於活化與不活化行為的偶合有所貢獻，尤其是與N端A3、V4、A5、G6、L7、Y8這些位置具有較大的關係。

The Shaker K+ channel is a member of the voltage-gated K+ channels which are composed of four subunits. Each subunit is a polypeptide which has six transmembrane segments（S1~S6）, with the C- and N-terminals both located intracellularly. The pore domain is composed of S5 and S6, whereas S4 has been considered as the voltage sensor which contains regularly spaced basic amino acids and may regulate the gating of the channel by sensing the change of membrane potential. The Shaker K+ channel tends to be inactivated after activation, and there are two mechanisms of inactivation：P/C type and N type inactivation. P/C type inactivation takes place more slowly than N type inactivation and may result from collapse of the outer part of the pore. On the other hand, N type inactivation is resulted from the inactivation ball which is probably formed by the N-terminal. In L366W, the existence of the inactivation ball alters the activation curve dramatically. L366R and L366K show slow inactivation kinetics and decreased voltage dependence of inactivation. We made point mutations and double mutations on L366 and N-ter inactivation ball to probe the possible interaction between the two parts of the channel. Point mutations on L7 and Y8 to polar amino acid abolish fast inactivation but the activation curve remains similar to WT. There are also remarkable differences between WT and point mutations on A3、V4、A5 and G6 to polar amino acid in fast inactivation, including rightward shift of Vh and increase of k value of the inactivation curve, decrease of inactivation rate, and increase of the ratio between sustained current and peak current. However, point mutations on G9、L10、G11 to polar amino acid affect fast inactivation relatively weakly. In L366D, the Vh of activation curve is shifted leftward by about 37 mV without significant changes in the k value. In contrast, the k value of the inactivation curve is almost doubled. For those point mutations of inactivation ball and L366D which decrease the coupling between activation and inactivation, L366D and mutations on position 3-8 of N-terminus（inactivation ball） to arginine may reconstitute the activation-inactivation coupling, respectively. The reconstitution of the coupling is demonstrated by the significaint modulation of the manoscopic, inactivation rates by membrane voltage（and thus the “crossover” of the inactivation phase at different voltages）, the decrease of the k value of inactivation curve and ratio between sustained and peak currents. We propose that there are direct interactions between the N-terminal inactivation ball（most likely A3 to Y8） and L366, possibly comprising an important molecular basis of the activation-inactivation coupling.

口試委員會審定書．．．．．．．．．．．．．．．．．．．．Ⅰ
誌謝．．．．．．．．．．．．．．．．．．．．．．．．．．Ⅱ
中文摘要．．．．．．．．．．．．．．．．．．．．．．．．Ⅲ
英文摘要．．．．．．．．．．．．．．．．．．．．．．．．Ⅳ
第一章 導論．．．．．．．．．．．．．．．．．．．．．．1
第二章 材料與方法．．．．．．．．．．．．．．．．．．．9
第三章 結果．．．．．．．．．．．．．．．．．．．．．．13
第四章 討論．．．．．．．．．．．．．．．．．．．．．．21
圗次
圖1 電位開關性鉀離子通道的topology及N端、S4的胺基酸序列 29
圖2 N端L7、Y8突變之電流紀錄．．．．．．．．．．．．．． 30
圖3 L7、Y8突變通道的活化曲線及活化速率．．．．．．．． 31
圖4 L366D突變通道的電流記錄與活化曲線及活化速率 ．．． 32
圖5 L366D突變通道的不活化曲線及不活化速率．．．．．．．33
圖6 L7DL366D、L7RL366D突變通道的活化曲線及time to Peak 34
圖7 L7DL366D、L7RL366D突變通道的不活化速率及sustained
current與peak current 比例．．．．．．．．．．． 35
圖8 Y8DL366D、Y8RL366D突變通道的活化曲線及活化速率及time
to peak．．．．．．．．．．． 36
圖9 Y8DL366D、Y8RL366D突變通道的不活化曲線、不活化速率及
sustained current和peak current比例．．．．． ． 37
圖10 A5R、G6R突變通道的活化、不活化曲線、活化速率及電流峰
值與殘餘電流的比例．．．．．．．．．．．．．．．38
圖11 A5D、G6D突變通道的電流記錄與活化曲線及活化速率．．39
圖12 G6DL366D、G6RL366D突變通道的活化曲線、time to peak40
圖13 G6DL366D、G6RL366D突變通道的不活化曲線及不活化速率及
電流峰值與殘餘電流的比例．．．．．．．．．．． 41
圖14 A5DL366D、A5RL366D突變通道的活化曲線、time to peak 42
圖15 A5DL366D、A5RL366D突變通道的不活化曲線及不活化速率43
圖16 A3R、V4R突變通道的活化、不活化曲線、活化速率及電流峰
值與殘餘電流的比例．．．．．．．．．．．．．．． 44
圖17 V4DL366D、V4RL366D突變通道的活化曲線、不活化曲線、
time to peak 及不活化速率．．．．．．．．．．．．45
圖18 A3DL366D、A3RL366D突變通道的活化曲線、time to peak46
圖19 A3DL366D、A3RL366D突變通道的不活化曲線、不活化速率及
Sustained current和peak current比例．．．．．． 47
圖20 G9R、L10R、L10D突變通道的活化曲線及time to peak．48
圖21 G9R、L10R、L10D突變通道的不活化曲線及不活化速率． 49
圖22 G9DL366D、G9RL366D突變通道的活化曲線、不活化曲線、
time to peak 及不活化速率．．．．．．．．．．．． 50
圖23 L10DL366D、L10RL366D突變通道的活化曲線、不活化曲線、
time to peak 及不活化速率．．．．．．．．．．．． 51
圖24 G11R、G11D、G11RL366D、G11DL366D突變之電流記錄．．52
圖25 G11D、G11R及其與L366D雙突變突變通道的活化曲線、
不活化曲線、time to peak不活化速率．．．．．．． 53
參考文獻．．．．．．．．．．．．．．．．．．．．．．． 54

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