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標題: | 鑑識與電位控制型鉀離子通道Kv4.3有交互作用的蛋白質 Identification and characterization of Kv4.3 interacting proteins |
作者: | 羅祥 Hsiang Lo |
指導教授: | 湯志永 Chih-Yung Tang |
關鍵字: | 酵母菌雙雜交系統,鉀離子通道, yeast two-hybrid,potassium channel,Kv4.3, |
出版年 : | 2018 |
學位: | 碩士 |
摘要: | 人類基因KCND3 encode出電位調控鉀離子通道Kv4.3,Kv4.3屬於Shal-type family (Kv4 family)次家族成員之一,廣泛表現在整個大腦、心臟及平滑肌當中,並且已有研究指出當病人KCND3基因發生突變時會導致SCA19/22脊髓小腦萎縮症;顯性體染色體遺傳性疾病,目前文獻指出當位於小腦的Purkinje cell中發生突變的Kv4.3 α subunit,會dominant negative影響wild type Kv4.3α subunit由ER送至細胞膜上表現;也會與wild type Kv4.3 α subunit進行聚合,並且影響通道的功能,但是對於Purkinje cell死亡機制目前尚不清楚,為了對Kv4.3有更進一步的認識,我們利用酵母雙雜交系統尋找與Kv4.3交互作用之蛋白質,希望能透過了解這些蛋白質在細胞中已知參與的機制、訊息傳導路徑,進一步推測對Kv4.3的調控性質。在對於大鼠腦部cDNA library進行篩選後,我們共發現了318個clonies可能與Kv4.3通道蛋白有交互作用,在經過進一步的序列分析和扣除胺基酸序列有frame shift後,剩下12個可能與Kv4.3通道蛋白有交互作用的蛋白質,我們從中挑選出來7個作為研究目標,進一步以實驗證明與Kv4.3通道蛋白交互作用的能力。首先我利用酵母雙雜交實驗 (包含X-gal測試分析與Leucine需求分析實驗)以釐清Kv4.3和其可能相互作用蛋白質之相關性。在酵母雙雜交實驗中,這12個蛋白質都能在進行X-gal測試分析實驗使菌落由白變藍,並且在缺乏白胺酸的培養皿上生長。我們進一步以GST pull down與Co-IP實驗之方式驗證,發現各有5個得到正反應。針對本篇研究所篩選到的蛋白質,我們除了以不同的生化實驗證明他們的交互作用外,我們期望將來能夠以更多生化實驗及電生理對這些蛋白質與Kv4.3之間的交互作用更深入的探討與分析,以了解Kv4.3在細胞內的各種生化機制。 Human gene KCND3 encode voltage-gated potassium channel Kv4.3. Kv4.3 highly express in brain, heart, and smooth muscle, which belong to Shal-type family (Kv4 family). Spinocerebellar ataxia type 19/22 (SCA19/22) is a dominantly inherited neurodegenerative, clinically heterogeneous disorder caused by mutations in KCND3 and Purkinje cell loss in the cerebellum consequently. SCA19/22mutant Kv4.3 subunits exerted dominant negative effects on WT Kv4.3 either in endoplasmic reticulum (ER)-Golgi membrane trafficking or reduced Kv4.3 channel functioning.In order to understand further about Kv4.3, we applied C-termius and N-terminus of Kv4.3 as the bait for yeast two-hybrid screening to search for novel Kv4.3 interacting proteins. 318 prey clonies were identified after screening a rat brain cDNA library. By X-gal assays, leucine requirement tests, yeast plasmid extract, colony PCR, sequencing and eliminating the clones with frame shit, we have obtained 12 potential candidate proteins. We chose 7 candidates for further characterization. GST pull down assay and Co-IP assay were performed to reconfirm the interaction between Kv4.3 channels and candidates. We found the five of the seven candidates have positive reaction in GST pull down assay; and found the five of the seven candidates have positive reaction in Co-IP assay. In this study, we found novel candidate proteins interacting with Kv4.3, and we performed experiments to prove it. We hope to better understand about physiological mechanism of Kv4.3 potassium channel with further biochemical experiment and electrophysiology experiments. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/79059 |
DOI: | 10.6342/NTU201802906 |
全文授權: | 未授權 |
電子全文公開日期: | 2023-10-11 |
顯示於系所單位: | 生理學科所 |
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