KChIP (Kv channel-interacting protein)為具有4 個EF-hands 的蛋白質已知可調控Kv channel 電生理功能，本論文主要探討KChIP2.2 與KChIP4a 和Kv4.2 channel 分子間結合的影響因子。在沒有鎂與鈣離子存在時，相較KChIP4a，KChIP2.2 與Kv channel 有明顯的結合反應，但兩者與Kv channel 分子間的結合可隨鈣離子及鎂離子濃度增加而增加，由突變蛋白和Kv channel 結合反應顯示KChIP4a 與KChIP2.2 和Kv channel 之結合作用無法由單一分子區域決定之。螢光實驗結果中發現KChIP2.2 對於鈣離子結合具有high affinity 與low affinity Ca2+-binding sites 而KChIP4a 僅只有一個low affinity Ca2+-binding site，但兩者僅具有一種Mg2+-binding site，由突變蛋白分析結果顯示KChIP2.2 之EF-hand 4 與其對鈣離子的high affinity site 有關，但是其他EF-hands 結構的完整性對於維持其對鎂與鈣離子之親和性也具有重要角色。與二價離子結合後KChIP2.2 的熱穩定性會降低，但KChIP4a
卻反而上升， Proteolytic digestion 與thiol reactivity 的實驗反應出KChIPs 結合鎂離子與鈣離子後構形產生不同的改變，在細胞內蛋白質分佈的實驗中可以增加KChIP2.2 分佈在細胞膜的情形但KChIP4a 則否，另外與老鼠腦萃取液中蛋白質的結合反應，也受到鈣離子之影響。綜合上述的結果，顯示KChIP2.2 與KChIP4a 和二價離子結合之後所誘導的結構變化，影響其和Kv channel 結合及細胞內的分佈扮演重要的角色

Kv channel interacting proteins (KChIPs) are Ca2+-binding proteins with four EF-hands and well-known to modulate Kv4.2 channel gating. The present study is carried out to investigate the molecular mechanism related to regulate the interaction of KChIP2.2 and KChIP4a with Kv channel. In comparison with KChIP4a, the interaction of KChIP2.2 with Kv4.2 was more obvious in the absence of Ca2+ or Mg2+. However the binding of KChIP2.2 and KChIP4a toward Kv4.2 increased with increasing Ca2+ and Mg2+ concentration. Nevertheless, no individual regions within KChIP2.2 and KChIP4a could exclusively fulfill the interaction between KChIPs mutants and Kv channel. Fluorescence measurement showed that KChIP2.2 possessed both high affinity and low affinity Ca2+-binding sites, but only low affinity Ca2+-binding site was observed with
KChIP4a. However, both of them have only one Mg2+-binding site. Studies on the truncated mutants revealed that the EF-hand 4 of KChIP2.2 was related to high affinity binding with Ca2+, and the integrity of molecular structure of KChIP2.2 and KChIP4a was important for Ca2+ -and Mg2+-binding. The thermal stability of KChIP2.2 and KChIP4 was found to be differentiately affected by Ca2+ and Mg2+. Proteolytic digestion and thiol reactivity assays also supported that Ca2+ and Mg2+-induced conformational change of KChIP2.2 was differed from KChIP4a. Moreover, in cells
co-transfected with Kv4.2 cDNA, it was formed that KChIP2.2 trafficking to the cell surface was increased by elevating intracellular Ca2+ concentration, but no noticeable change was observed for KChIP4a. Taken together, these results suggest that the conformational changes of KChIP2.2 and KChIP4a differently induced by Ca2+ and Mg2+ affect their binding with Kv channel and/or cellular distribution.

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