糖基化对Ca_v3.2 T型钙通道的作用
发布时间：2018-07-14 16:01
【摘要】：目的:糖基化是蛋白质翻译后修饰的重要方式之一,并在维持蛋白质的正常结构及调节蛋白质的功能中都发挥着重要作用。本研究利用衣霉素阻断N型糖基化,并使用全细胞膜片钳技术探究阻断糖基化后的Ca_v3.2-T型钙通道的改变,以期了解糖基化更多的生理病理作用。方法:1细胞培养本研究所用细胞为转染人类Ca_v3.2-T型钙通道基因的HEK293细胞。2电生理技术在室温条件下使用全细胞膜片钳技术记录HEK细胞上的Ca_v3.2-T型钙离子通道电流。稳态激活测定设计为:维持电位为-100mV,测试电位为-100mV至+50mV,阶跃电压5mV,步宽300ms,刺激间隔为2s。稳态失活测定设计为:维持电位为-100mV,预刺激为-100mV至-20mV,阶跃电压为5mV,测试电位为-20mV,步宽300ms,刺激间隔2s。结果:衣霉素对Ca_v3.2-T型钙通道电流的抑制作用非常显著,并具有时间关联性。衣霉素(10mg/L)处理细胞1h及6h后,电流未发生改变,12h后,实验组电流约为对照组电流一半,而在24h后,抑制效果进一步增强。1h组对照组与实验组电流密度分别为40.98±3.98pA/pF(n=14)、-40.70±3.26pA/pF(n=12,P=0.957)。6h组对照组与实验组电流密度分别为-41.22±3.78pA/pF(n=18)、-41.81±3.96pA/pF(n=15,P=0.916)。12h组对照组与实验组电流密度分别为41.00±4.26pA/pF(n=11)、18.54±2.68pA/pF(n=9,P0.001)。24h组对照组与实验组电流密度分别为-44.32±1.91pA/pF(n=12)与13.06±1.59pA/pF(n=17,P0.001)与电流相一致,衣霉素导致Ca_v3.2-T型钙通道稳态激活曲线右移,并也呈现相似的时间相关性。12h时,稳态激活曲线开始出现右移,24h时偏移进一步增加加剧。1h组对照组及实验组半激活电位为-44.24±1.28mV(n=14)和-44.83±2.01mV(n=12,P=0.799)。6h组对照组及实验组半激活电位为-44.15±0.386mV(n=18)和-43.70±0.493mV(n=15,P=0.470)。而12h时,稳态激活曲线开始出现右移,半激活电位由44.76±0.99mV(n=11)偏移至42.17±0.53mV(n=9,P=0.045)。而24h时偏移进一步增加加剧,由半激活电位由44.13±1.03mV(n=12)偏移至39.09±0.65mV(n=17,P0.001)。而与此同时,稳态失活曲线没有发生明显改变。结论:衣霉素对Ca_v3.2-T型钙通道电流的抑制作用呈现明显的时间相关性。此外衣霉素阻断N型糖基化可以导致Ca_v3.2-T型钙通道激活曲线右移,但不影响稳态失活曲线,这将导致Ca_v3.2-T型钙通道窗口电流减小。这些改变会显著影响T型钙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. 鎶戝埗鏁堟灉杩涗竴姝ュ寮，
本文编号：2122176
[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. 鎶戝埗鏁堟灉杩涗竴姝ュ寮，
本文编号：2122176
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