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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
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dc.contributor.advisor | 樓國隆(Kuo-Long Lou) | |
dc.contributor.author | Chun-Hung Kuo | en |
dc.contributor.author | 郭俊宏 | zh_TW |
dc.date.accessioned | 2021-06-13T05:51:53Z | - |
dc.date.available | 2006-08-03 | |
dc.date.copyright | 2006-08-03 | |
dc.date.issued | 2006 | |
dc.date.submitted | 2006-07-05 | |
dc.identifier.citation | Antonello Bonci, William A. Carlezon Jr., Ion channels and intracellular signaling proteins as potential targets for novel therapeutics for addictive and depressive disorders, Pharmacology & Therapeutics, 108, 65 – 75 (2005).
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/34023 | - |
dc.description.abstract | 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。 | zh_TW |
dc.description.abstract | 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. | en |
dc.description.provenance | Made available in DSpace on 2021-06-13T05:51:53Z (GMT). No. of bitstreams: 1 ntu-95-R93450011-1.pdf: 4785546 bytes, checksum: 3e5e9438d6e01f1e0d7a1c2f43e7e781 (MD5) Previous issue date: 2006 | en |
dc.description.tableofcontents | 目錄 2
表目錄 5 圖目錄 6 中文摘要 8 英文摘要 10 壹、序論 一、離子通道 13 二、鉀離子通道 14 三、內向型鉀離子通道 16 四、內向性鉀離子通道第一型 18 貳、實驗材料與儀器 一、實驗材料 22 二、實驗試劑與材料 22 三、實驗儀器 25 參、實驗方法 Kir1.1胞內區域蛋白質之表現 一、放大菌量 28 二、誘導產生目標蛋白 28 三、以聚丙烯醯胺電泳(SDS-PAGE)分析誘導後之蛋白質 29 四、膠片染色法 30 五、膠片乾燥法 31 六、蛋白質轉印 31 七、西方墨點法 31 八、透析 32 九、FPLC Protein Purify System純化目標蛋白 33 十、蛋白質定量 34 十一、以Thrombin對GST-融合蛋白進行切割 35 肆、實驗結果與討論 一、Kir1.1胞內區域蛋白質之表現 37 二、利用FPLC純化目標蛋白 38 三、對純化所得之融合蛋白進行蛋白質定量 39 四、利用Thrombin對C端GST-融合蛋白進行切割 39 伍、未來展望 40 陸、實驗圖表 43 柒、參考文獻 63 | |
dc.language.iso | zh-TW | |
dc.title | 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 | zh_TW |
dc.title | Expression and Purification of Intracellular Domains
of Mammalian Inwardly Rectifying Potassium Channel Kir1.1 | en |
dc.type | Thesis | |
dc.date.schoolyear | 94-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 周綠蘋,劉宏輝,廖彥銓,張百恩 | |
dc.subject.keyword | 鉀離子通道,內向性性整流,蛋白質純化, | zh_TW |
dc.subject.keyword | potassium channel,inwardly rectifying,protein purification, | en |
dc.relation.page | 75 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2006-07-05 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 口腔生物科學研究所 | zh_TW |
顯示於系所單位: | 口腔生物科學研究所 |
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