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研究生:陳敬哲
研究生(外文):Jing-Jer
論文名稱:以去磷酸化酶抑制劑與PMA探討KCNQ4鉀離子通道之功能性研究
論文名稱(外文):Functional study of phosphotase inhibitors and PMA on KCNQ4 potassium channel
指導教授:林明忠林明忠引用關係
指導教授(外文):Min-Jon Lin
學位類別:碩士
校院名稱:中山醫學大學
系所名稱:生物醫學科學學系碩士班
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2010
畢業學年度:98
語文別:中文
論文頁數:59
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我們想了解KCNQ4鉀離子通道與磷酸化之間的相關性為何,而選擇使用HEK293t cell與Xenopus laevis oocytes 兩種離子通道表達系統,分別使用全細胞電位箝定技術與雙電極電位箝定技術記錄表達的KCNQ4鉀離子通道功能,並給予引發或抑制細胞內磷酸化之適當藥劑處理,以探討KCNQ4鉀離子通道與磷酸化的相關性,並藉由生物資訊學的磷酸化胺基酸位點預測軟體協助下,將KCNQ4鉀離子通道C端上可能的磷酸化胺基酸位點,以位置導向點突變技術加以改變,嘗試尋找可能的磷酸化胺基酸位點,並比較KCNQ4鉀離子通道表達於HEK293t cell與Xenopus laevis oocytes 兩種表達系統之差異。由我們的實驗結果推論可能由於Xenopus laevis oocytes與HEK293t cell其細胞內生性磷酸化程度不同,而表達於HEK293t cell上之KCNQ4鉀離子通道受較高程度的磷酸化作用,因此離子通道活化曲線在PMA與去磷酸化抑制劑作用下不產生偏移,然而鉀離子電流的抑制現象卻也提示了,PMA與去磷酸化酶抑制劑可能存在磷酸化調控方式以外的其他直接或間接的離子通道調控方式。在經由KCNQ家族蛋白多重序列比對之後,我們發現在S494A附近有一內質網輸送訊號(RXR motif),或許由於S494A的突變引起此區域結構的改變,而導致無電流的結果。

We would like to understand the relationship between KCNQ4 potassium channel and phosphorylation. HEK293t cell and Xenopous laevis oocyte ion channel expression systems would be our choice. To use two electrodes voltage clamp (TEVC) and whole cell voltage clamp recording KCNQ4 potassium current which expressed on Xenopous laevis oocyte and HEK293t cell, and treating with phosphorylation chemicals to increase or decrease phospohryaltion state. On the other hand, we predict the possible phosphorylation site of KCNQ4 potassium channel by Bioimformatics phosphorylation site prediction software. The phosphoryaltion site prediction is focused on the C-terminal of KCNQ4, and use site-directed point mutation to change amino acid on possible phosphorylation sites. Our results conclude that there are different phosphorlyation states on Xenopous laevis oocyte and HEK293t cell. KCNQ4 express on HEK293t cell has higher phosphorylation state than Xenopous laevis oocyte, so PMA does not shift the V1/2 curve. However, the KCNQ4 current reduction results reveal that there could be another KCNQ4 regulation mechanism differ from phosphorylation. After KCNQ family protein sequence multialigment, we find a ER trafficking signal (RXR motif) which is near the S494A. S494A possibly chages a part of ER trafficking signal, and cuase the no current result.

中文摘要…………………………………………………………………1
Abstract…………………………………………………………………2
序論(Introduction)……………………………………………………3
材料與方法(Materials and Methods)…………………………………9
一.分子生物學(Molecular biology)
KCNQ4-pTLN質體來源(Resource of plasmid KCNQ4-pTLN)
KCNQ4-pcDNA3.1質體建構(Construction of plasmid KCNQ4-pcDNA3.1)
DH5α大腸桿菌之轉型(Transformation)
質體純化(Plasmid purification)
DNA膠體電泳(Gel electrophoresis)
cRNA的製備(In vitro transcription)
位置導向點突變技術(Site directed point mutation)
二.細胞生物學(Cell biology)
HEK293t細胞株培養(HEK293t cell line culture)
轉染(Transfection)
非洲爪蟾卵母細胞的製備(Preparation of Africa Xenopus oocytes)
cRNA顯微注射(cRNA micro-injection)
三.電生理學(Electrophysiology)
雙電極電位箝定技術(Two Electordes Voltage Clamp;TEVC)
全細胞箝定技術(Whole cell patch-clamp)
四.生物資訊學(Bioinformatics)
人類KCNQ4磷酸化位點預測(Phosphorylation site prediction of KCNQ4)
五.資料分析(Data analysis)
六.藥劑(Drug)
結果(Results)…………………………………………………………27
非洲爪蟾卵細胞異源性表達KCNQ4鉀離子通道電流的特徵……………………27
去磷酸化酶抑制劑Calyculin A與Cyclosporin A對KCNQ4鉀離子通道於爪蟾卵細胞異源性表達鉀離子電流的作用………………………………………………28
KCNQ4經轉染於HEK293t cell上表達之鉀離子通道電流特徵…………………29
PKC活化劑PMA對表達於HEK293t細胞上KCNQ4鉀離子電流之影響………31
去磷酸化酶抑制劑Okadaic acid、Calyclulin A與Cyclosporin A對表達於HEK293t細胞上KCNQ4鉀離子電流之影響…………………………………………………32
KCNQ4蛋白質C端磷酸化胺基酸位點預測與位置導向點突變結果……………33
討論(Discussion)……………………………………………………34
Xenopus oocytes與HEK293t細胞株離子通道表達系統之差異…………………34
磷酸化位點預測與位置導向點突變結果無明顯差異……………………………35
S494A胺基酸點突變導致KCNQ4失去功能………………………………………35
圖像(Figure)…………………………………………………………37
Figure 1:爪蟾卵細胞經雙電極電位箝定所記錄之離子通道電流。……………37
Figure 2:KCNQ4鉀離子通道表達於非洲爪蟾卵細胞之電位功能性分析。……………………………………………………………………………………39
Figure 3:KCNQ4鉀離子通道於爪蟾卵細胞異源性表達並以去磷酸化酶抑制劑處理之電流與電位依賴性曲線圖。……………………………………………………40
Figure 4:HEK293t細胞株經Lipofectamin 2000轉染KCNQ4/EGFP(10:1)。……42
Figure 5:HEK293t細胞經全細胞電位箝定技術所記錄之離子通道電流。………43
Figure 6:KCNQ4鉀離子通道表達於HEK293t細胞株之電位功能性分析。……………………………………………………………………………………45
Figure 7:PKC活化劑PMA作用下對KCNQ4鉀離子通道之影響。……………………………………………………………………………………46
Figure 8:去磷酸化酶抑制劑作用下對HEK293t細胞表達KCNQ4鉀離子通道電流影響。………………………………………………………………………………48
Figure 9:以生物資訊學方法預測可能的磷酸化位點與KCNQ4鉀離子電流記錄結果。…………………………………………………………………………………50
參考文獻(Reference)…………………………………………………52
附件
附件一 KCNQ4 protein structure…………………………………………………57
附件二 Site-directed point mutation primer sequences……………………58
附件三 KCNQ family S494A protein sequence multialigment………………59


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