摘 要
在先前的研究中，以yeast-two hybrid方法得知

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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