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

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.