Potassium channel-interacting protein 1 (KChIP1) 為一調控 A-type potassium channels (Kv4) 功能並增加其細胞表面表現量的neuron calcium sensor (NCS) proteins。先前研究認為 KChIP1 可透過 N 端 myristoyl group 結合至細胞膜，但卻不夠至穩定蛋白質與膜之間的結合，因此本論文將探討是否有其他結構區域會影響 KChIP1 與細胞膜之結合。 首先以 hydropathy profile 分析得知第三及第四個 EF-hands 為較疏水的區域。 在去除此區域後不僅造成 Trp 微環境結構的改變，也明顯減少 KChIP1 之疏水性，但其二級結構仍可維持 α-helix 構型。 而 truncated KChIP1 也對酯質 (lipid) 之結合能力有顯著下降並因此影響其在細胞內的 membrane localization，推測 EF-hands 3 及 4 具有增進 KChIP1 與細胞膜結合的能力。 此外，truncated KChIP1 也喪失對 phosphatidylserine 結合之特異性並降低兩者之間的親和力。 根據 FTIR spectra 結果顯示隨著酯質 large unilamellar vesicles 中 phosphatidylserine 比例上升，結合上酯質之 KChIP1 其 α-helix 的結構也有隨之改變的趨勢。 且從蛋白質聚合反應得知，phosphatidylserine 與 Kv4 會促進 KChIP1 形成 tetramer 來聚集在酯質上，另外與 Kv4 結合會增加 KChIP1 與酯質結合的能力。 綜合上述結果得知，EF-hands 3 及 4 motifs 影響 KChIP1 與細胞膜結合時之親和力及特異性，並在 KChIP1 展現生理功能上可能扮演重要的角色。

Potassium channel-interacting protein 1 (KChIP1), a Ca²⁺ sensor protein, regulates the function of A-type Kv4 potassium channels and increases their cell surface expression. Myristoylation at the N-terminus of KChIP1 has been suggested to facilitate membrane-binding, but was not sufficient for stable membrane assaociation. The aim of the present study is to investigate whether EF-hand motifs of KChIP1 are crucial for membranal targeting in addition to the N-terminal myristoyl group, and how the membrane association of KChIP1 is influenced by lipid compositions. According to hydropathy profile, EF-hands 3 and 4 of KChIP1 showed highly hydrophobicity. After deleting EF-hands 3 and 4, the altered microenvironment of Trp residue and decreased hydrophobicity were found in truncated KChIP1, but it still maintained α-helix structure. Furthermore, truncated KChIP1 exhibited lower lipid-binding ability, affecting intracellular membrane localization and was almost diminished underlying increasing membrane permeability by digitonin in cells, suggesting that intact EF-hands 3 and 4 may be related to the anchorage of KChIP1 on cellular membrane. KChIP1, but not mutant, specifically bound with phosphatidylserine by lipid binding assay and the FTIR spectra showed the change of α-helix structure by binding lipid large unilamellar vesicles was dependent on phosphatidylserine. Either phosphatidylserine or potassium channels enhanced KChIP1 to form tetramer for targeting to phospholipids by using chemical cross-linking assay. Taken together, our data highly suggest that intact of EF-hands 3 and 4 should structurally and functionally involve in fulfilling the physiological activity of KChIP1.

目錄 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
中文摘要 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
英文摘要 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
縮寫表 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
緒論 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
材料 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .11
實驗方法 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
結果 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
討論 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
圖表 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
參考文獻 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43

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