請用此 Handle URI 來引用此文件:
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/34023
標題: | 哺乳類動物之內向性整流型鉀離子通道第一型胞內區域蛋白質之表現與大量純化 Expression and Purification of Intracellular Domains of Mammalian Inwardly Rectifying Potassium Channel Kir1.1 |
作者: | Chun-Hung Kuo 郭俊宏 |
指導教授: | 樓國隆(Kuo-Long Lou) |
關鍵字: | 鉀離子通道,內向性性整流,蛋白質純化, potassium channel,inwardly rectifying,protein purification, |
出版年 : | 2006 |
學位: | 碩士 |
摘要: | 內向性整流型鉀離子通道Kir1.1 (Inwardly Rectifying Potassium Channel; Kir1.1)為鉀離子通道的一種,其特性為當細胞膜電位低於平衡電位時,能允許胞外的鉀離子流入細胞內;而當膜電位高於平衡電位時,則傾向關閉不讓鉀離子流出細胞外。此類型鉀離子通道的生理功能主要在維持細胞靜止膜電位、神經突觸的興奮性、以及腎小管中鉀的再吸收等。此類通道之異常可導致如Bartter’s syndrome等疾病的產生。
已知Kir1.1 鉀離子通道之磷酸與去磷酸化的過程調控著離子通道的活化,近年來的研究也指出Kir1.1 鉀離子通道之磷酸化的調控是透過蛋白質激酶A (cyclic AMP-dependent protein kinase)的作用並藉由PIP2的參與而產生。另外Kir1.1 鉀離子通道也被證明會受到細胞內酸鹼值(pH i)之影響:當細胞內酸鹼值下降時,會引起Kir1.1 通道的關閉。目前推測這些造成Kir1.1 通道開閉的情形可能牽涉到膜內區域構形的重組,然而其細部的機制卻不清楚。因此我們企圖藉由瞭解Kir 1.1 通道N 端及C 端膜內區域的蛋白結構來釐清上述機制。 在本篇論文中我們成功地在E. coli 中表現出Kir1.1 鉀離子通道的 N 端及C 端膜內區域的融合蛋白(Fusion Protein),也藉由親9和性管柱純化出C 端的蛋白質,然而N 端的蛋白質因表現量太少尚未純化出來。未來我們將朝著大量表現N 端蛋白的近程目標以及結晶並解出N 端C 端蛋白的3D 結構的遠程目標來邁進。 As a member of the family of inwardly rectifying potassium channels, Kir1.1 (ROMK1) channel is widely distributed in different tissues and regulates many important physiological functions, including maintenance of resting potential, synaptic excitability, and renal K+ transport. Phosphorylation and dephosphorylation are believed to regulate the activation of Kir1.1 channels. Recently, it was demonstrated that the phosphorylation of Kir1.1 channels is mediated by cyclic AMP-dependent protein kinase (PKA) through the participation of PIP2. In addition, it has been since long proven that decrease in intracellular pH (pHi) can result in the channel closure of Kir1.1. The molecular mechanism responsible for such regulations may involve the structural rearrangements of the intracellular N- and C-terminal domains in Kir1.1 channels. However, the details required for further interpretation regarding this remain unclear. Therefore, we are in attempts to solve this problem through the determination of 3-D structures of these domains in Kir1.1 channels. To reach this goal, in this study, we have successfully expressed the N- and C-terminal intracellular domains of Kir1.1 in E. coli as fusion proteins containing GST-tag. The C-terminal domain protein has been obtained through efficient purification with affinity chromatography, whereas, however, the N-terminal part was still unavailable due to the limited expression level. Our future studies will be focused in the large-scale expression and purification of the Kir1.1 N-terminal domain protein as short-term goal with respect to the crystallization and structural determination for both N- and C-terminal parts in a long run. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/34023 |
全文授權: | 有償授權 |
顯示於系所單位: | 口腔生物科學研究所 |
文件中的檔案:
檔案 | 大小 | 格式 | |
---|---|---|---|
ntu-95-1.pdf 目前未授權公開取用 | 4.67 MB | Adobe PDF |
系統中的文件,除了特別指名其著作權條款之外,均受到著作權保護,並且保留所有的權利。