臺灣博碩士論文加值系統

摘 要
在先前的研究中，以yeast-two hybrid方法得知 ��-bungarotoxin (beta-bgt)的B chain會與老鼠腦中的KChIP3產生分子間的交互作用。KChIP (Kv channel-interacting protein)鉀離子通道結合蛋白為電位驅動型鉀離子通道 (Voltage-gated potassium channel, Kv)其中之Type-A potassium channel (Kv4)之輔助結合蛋白，並調控其功能。 beta-bgt純化自台灣雨傘節蛇毒，由分子間双硫鍵鍵結A chain及B chain兩個subunit而成，屬於突觸前神經毒素。 由pull down結果顯示，KChIP1~4蛋白質中僅有KChIP3會與 beta-bgt結合並受到鈣離子的調節，且結合位置分別是以KChIP3上第三及第四個EF-hand區域結合 beta-bgt。同時，也由老鼠腦組織蛋白中找到會與 beta-bgt結合的KChIP3蛋白，於是推論KChIP3可能是 beta-bgt在細胞內的目標結合蛋白之一。 此外，也以pull-down assay分析可能會與KChIP結合的其他蛇毒蛋白，發現台灣眼鏡蛇Cardiotoxin3 (CTX3)蛇毒蛋白能與KChIPs產生分子間結合作用。 由cross-linking實驗發現CTX3與KChIP1為1: 1的比例結合，也藉由pull down實驗證明KChIP1上的第三、第四個EF-hand去除後，即失去CTX3的結合能力，但KChIP3與KChIP1不同的是，氮端、第四個EF-hand去除以及同時去除第三和第四個EF-hand的KChIP3 mutants皆能和CTX3結合。 此外，實驗證明CTX3與KChIPs間的結合是Ca2+-independent，也由螢光分析得知CTX3在鈣離子存在下，會降低KChIP1與ANS螢光物質的結合，但在KChIP1與Kv4的結合中，CTX3卻扮演一個異位調節的角色，能增強兩分子間的作用。 由於KChIP蛋白結合Kv4後會進一步調節其功能，也因此藉由beta-bgt與CTX3結合KChIP並調節KChIP與Kv4結合作用的特性，當作模板做為藥物設計之基礎，應用於調控鉀離子通道的生物活性。

Abstract
Our previous study showed that KChIP3 (Kv Channel Interacting Protein 3) probably was a physiological targete protein of beta-bungarotoxin (beta-Bgt) as evidenced by yeast two-hybrid system. Thus, extensive efforts are carried out to explore the molecular interaction between KChIP3 and beta-Bgt in the present study. KChIPs are potassium (Kv) channel-interacting proteins that bind to the 1~90 amino acid of N-terminus of Kv4 alpha-subunits and regulate the ion current density, shift the voltage dependence of activation and speed their recovery from inactivation. beta-Bgt, a presynaptic neurotoxin purified from Bungarus multicinctus venom, consists of A chain and B chain which cross-linked by an interchain disulfide bond. The results of pull-down assay revealed that, in contrast to other KChIP proteins, KChIP3 bound with beta-Bgt. Moreover, it was found that the B chain of beta-Bgt was a functional subunit in the binding with KChIP3, and the binding of KChIP3 to beta-Bgt showed a Ca2+-dependent manner. Removal of the third and the fourth EF-hand regions of KChIP3 abolished its interaction with beta-Bgt. Noticeably, the binding of beta-Bgt with KChIP3 did not influence the interaction between KChIP3 and Kv4. In the meantime, rat brain KChIP3 could be isolated using a beta-Bgt-Sepharose column. These observations suggest that KChIP3 is an intra-cellular target recognized by beta-Bgt. Accidently, it was found that direct protein-protein interaction between Taiwan cobra cardiotoxin3 (CTX3) and potassium channel-interacting proteins (KChIPs) was investigated. It was found that KChIPs bound with CTX3, in which KChIP1 and CTX3 formed a 1:1 complex as evidenced by the results of chemical crosslinking. Pull-down assay revealed that the intact EF-hand 3 and 4 of KChIP1 was critical for CTX3-binding. Whereas, all mutated KChIP3 were able to bind with CTX3. In contrast to the interaction between KChIP1 and KvN, the binding of CTX3 to KChIP1 showed a Ca2+-independent manner. Fluorescence measurement revealed that CTX3 affected the binding of ANS to Ca2+-bound KChIP1, but not Ca2+-free KChIP1. Alternatively, KChIP1 simultaneously bound with KvN and CTX3, and the interaction between KChIP1 and KvN was enhanced by CTX3. In terms of the fact that KChIPs regulate the electrophysiological properties of Kv K+ channel, the potentiality of beta-bgt and CTX for this biomedical application could be considered.

目 錄


中文摘要 ----------------------------------- 1
英文摘要 ----------------------------------- 2
緒論 --------------------------------------- 4
縮寫 --------------------------------------- 9
實驗材料 ----------------------------------- 10
實驗方法 ----------------------------------- 12
實驗結果 ----------------------------------- 25
討論 --------------------------------------- 34
圖表 --------------------------------------- 40
參考文獻 ----------------------------------- 62
附錄 --------------------------------------- 69

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