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  • 學位論文

KCNQ4 開啟劑衍伸物對於 Kv7.4 鉀離子電流的影響

Studies of the novel KCNQ4 opener analogues on Kv7.4 potassium currents.

指導教授 : 林明忠

摘要


鉀離子通道在哺乳動物組織內是分布最廣泛的離子通道。鉀離子通道在神經元與肌肉細胞的細胞膜上扮演穩定電位的重要角色。若突變則可能導致運動失調、癲癇等。 鉀離子通道可由Kv7 家族內的 KCNQ 蛋白組成。KCNQ分為 KCNQ1~KCNQ5,其中KCNQ4與聽覺系統有關。本論文主要針對 Kv7 家族內的 Kv7.4 (KCNQ4) 進行研究,此蛋白主要表現於內耳、骨骼肌、心肌內等,其異常可能導致先天性非症候群聽障 (DFNA2)、癲癇,與高血壓等相關疾病。目前研究已知 ML213、Retigabine 為鉀離子開啟劑。Retigabine 在臨床上應用於治療癲癇、疼痛與心律不整。本研究希望找出可以促進KCNQ4鉀離子通道開啟的最簡易結構,我們藉由修飾 Retigabine 和 ML213的結構合成新的十種化合物。實驗藉由使用人類胚胎腎細胞 (HEK293t) 大量表現 KCNQ4蛋白,再經全細胞電位箝定技術 (Whole cell patch clamp) 記錄給予新合成藥物對於鉀離子電流的變化。 研究結果顯示投與 Retigabine (10 μM)、ML213 (10 μM) 作為 positive control與合成結構相似物 #3070 (10 μM)、#0335(10 μM)、#1848(10 μM)、#0298(10 μM)、 #5907 (10 μM)、 #6696 (10 μM)、5540 (10 μM)、 #8692 (10 μM) 等藥物,將處理後的細胞進行電流分析。實驗得知經過這些藥物處理後,大部分的藥物發現KCNQ4鉀離子電流相較於控制組皆顯示上升的情況,將結果標準化後明顯發現,活化曲線向左移。其中又以 #3070最為顯著。然而投予 #0321(10 μM)、#0331(10 μM)、 #1574 (10 μM)、 #3839(10 μM)、#7294(10 μM)、#5002(10 μM) 對於KCNQ4鉀離子通道並沒有太大的影響。此外,投予 ML213 結構相似物,#0342 (10 μM),處理過後的細胞,其電流相較於控制組顯示下降的情況。 由本實驗結果顯示相較於其他十三種化合物,#5907、 #5540促進 KCNQ4鉀離子通道開啟的效果較為明顯,未來可以針對此化合物結構更進一步的探討研究。

並列摘要


K+ channels are the most widely distributed ion channels in mammals and play an important role in stabilizing membrane potential in neurons and muscle cells. Mutation of K+ channels may result in ataxia, epilepsy. In the present study, we mainly focus on the function of KCNQ4 (Kv7.4) protein. There are five members of the KCNQ family, which are reffered to as either KCNQ1-KCNQ5. KCNQ4 is related to auditory system. KCNQ4 is expressed in inner ears, skeletal muscle and cardiac muscle. We are already known that ML213 and retigabine are the KCNQ4 openers. Clinically, retigabine has been treated with epilepsy, pain and arrhythmia. To identify the common structure, we designed the novel Kv7.4 channel openers. We used human embryo kidney cells (Hek293) to express KCNQ4, and recorded K+ currents before and after treated with new analogues by whole cell patch clamp technique.The preliminary results revealed that the amplitude of KCNQ4 K+ currents are increased by treated with retigabine (10μΜ), ML213(10μΜ), #3070(10μΜ), #0335(10 μM), #1848(10 μM) #0298(10μΜ), #5907 (10 μM), #6696 (10 μM), 5540 (10 μM) and #8692 (10 μM). Furthermore we can find the activation curve was shifted toward less negative potentials by the treatment with KCNQ4 opener analogues (Retigabine, ML213, #3070, #1848, #0298, #0335, #5907, #6696, #5540 and #8692). Among these compounds, the #5907 and #5540 showed the most effective on KCNQ4 current. There are no effects on KCNQ4 with #3839(10μΜ), #1574(10 μM), #7294(10μΜ), #0321(10μΜ), #0331(10μΜ) and #5002(10μΜ). However the treatment with #0342 (10μΜ) causes the current amplitude decrease significantly. In conclusion, the KCNQ4 opener analogue, #5907 and #5540 showed more significant effect than the other nine compounds. Maybe we can further investigate the compound in the future.

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