【摘要】：目的:糖基化是蛋白質(zhì)翻譯后修飾的重要方式之一,并在維持蛋白質(zhì)的正常結(jié)構(gòu)及調(diào)節(jié)蛋白質(zhì)的功能中都發(fā)揮著重要作用。本研究利用衣霉素阻斷N型糖基化,并使用全細(xì)胞膜片鉗技術(shù)探究阻斷糖基化后的Ca_v3.2-T型鈣通道的改變,以期了解糖基化更多的生理病理作用。方法:1細(xì)胞培養(yǎng)本研究所用細(xì)胞為轉(zhuǎn)染人類Ca_v3.2-T型鈣通道基因的HEK293細(xì)胞。2電生理技術(shù)在室溫條件下使用全細(xì)胞膜片鉗技術(shù)記錄HEK細(xì)胞上的Ca_v3.2-T型鈣離子通道電流。穩(wěn)態(tài)激活測(cè)定設(shè)計(jì)為:維持電位為-100mV,測(cè)試電位為-100mV至+50mV,階躍電壓5mV,步寬300ms,刺激間隔為2s。穩(wěn)態(tài)失活測(cè)定設(shè)計(jì)為:維持電位為-100mV,預(yù)刺激為-100mV至-20mV,階躍電壓為5mV,測(cè)試電位為-20mV,步寬300ms,刺激間隔2s。結(jié)果:衣霉素對(duì)Ca_v3.2-T型鈣通道電流的抑制作用非常顯著,并具有時(shí)間關(guān)聯(lián)性。衣霉素(10mg/L)處理細(xì)胞1h及6h后,電流未發(fā)生改變,12h后,實(shí)驗(yàn)組電流約為對(duì)照組電流一半,而在24h后,抑制效果進(jìn)一步增強(qiáng)。1h組對(duì)照組與實(shí)驗(yàn)組電流密度分別為40.98±3.98pA/pF(n=14)、-40.70±3.26pA/pF(n=12,P=0.957)。6h組對(duì)照組與實(shí)驗(yàn)組電流密度分別為-41.22±3.78pA/pF(n=18)、-41.81±3.96pA/pF(n=15,P=0.916)。12h組對(duì)照組與實(shí)驗(yàn)組電流密度分別為41.00±4.26pA/pF(n=11)、18.54±2.68pA/pF(n=9,P0.001)。24h組對(duì)照組與實(shí)驗(yàn)組電流密度分別為-44.32±1.91pA/pF(n=12)與13.06±1.59pA/pF(n=17,P0.001)與電流相一致,衣霉素導(dǎo)致Ca_v3.2-T型鈣通道穩(wěn)態(tài)激活曲線右移,并也呈現(xiàn)相似的時(shí)間相關(guān)性。12h時(shí),穩(wěn)態(tài)激活曲線開始出現(xiàn)右移,24h時(shí)偏移進(jìn)一步增加加劇。1h組對(duì)照組及實(shí)驗(yàn)組半激活電位為-44.24±1.28mV(n=14)和-44.83±2.01mV(n=12,P=0.799)。6h組對(duì)照組及實(shí)驗(yàn)組半激活電位為-44.15±0.386mV(n=18)和-43.70±0.493mV(n=15,P=0.470)。而12h時(shí),穩(wěn)態(tài)激活曲線開始出現(xiàn)右移,半激活電位由44.76±0.99mV(n=11)偏移至42.17±0.53mV(n=9,P=0.045)。而24h時(shí)偏移進(jìn)一步增加加劇,由半激活電位由44.13±1.03mV(n=12)偏移至39.09±0.65mV(n=17,P0.001)。而與此同時(shí),穩(wěn)態(tài)失活曲線沒有發(fā)生明顯改變。結(jié)論:衣霉素對(duì)Ca_v3.2-T型鈣通道電流的抑制作用呈現(xiàn)明顯的時(shí)間相關(guān)性。此外衣霉素阻斷N型糖基化可以導(dǎo)致Ca_v3.2-T型鈣通道激活曲線右移,但不影響穩(wěn)態(tài)失活曲線,這將導(dǎo)致Ca_v3.2-T型鈣通道窗口電流減小。這些改變會(huì)顯著影響T型鈣通道的生理病理功能。
[Abstract]:Aim: glycosylation is one of the most important methods of protein posttranslational modification and plays an important role in maintaining the normal structure of protein and regulating the function of protein. In order to understand the physiological and pathological effects of glycosylation, the N-type glycosylation was blocked by itamin and whole-cell patch clamp technique was used to explore the changes of CaV3.2-T calcium channel after blocking glycosylated CaV3.2-T channel in order to understand the physiological and pathological effects of glycosylation. Methods HeK293 cells transfected with Cav3.2-T calcium channel gene were used to record Cav3.2-T calcium channel currents in HEK cells by whole-cell patch clamp technique at room temperature. The steady-state activation test was designed as follows: the maintenance potential was -100mV, the test potential was -100mV to 50mV, the step voltage was 5mV, the step width was 300msand the stimulus interval was 2s. The design of steady-state inactivation was as follows: maintenance potential was -100mV, prestimulation was -100mV to -20mV, step voltage was 5mV, test potential was -20mV, step width was 300msand stimulation interval was 2s. Results: the inhibitory effect of chlortetracycline on CaV3.2-T calcium channel current was significant and time-dependent. After treated with 10 mg / L for 1 h and 6 h, the current of the experimental group was about half that of the control group, but after 24 h, the current of the experimental group was about half that of the control group. 鎶戝埗鏁堟灉榪涗竴姝ュ寮，

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