非洲爪蟾卵母细胞表达系统中N-型电压门控钙离子通道新药筛选模型的建立与优化

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中文摘要：N-型电压门控钙离子(Ca2+)通道(N-type voltage-gated calcium channel，N-type VGCC，CaV2。2)在突触前末端响应动作电位介导Ca2+内流，并在突触发生、神经递质释放和伤害性信号传递中发挥重要作用，是神经痛(慢性痛)等重大疾病治疗药物研发的关键靶点。由于钙离子通道体外表达困难，通道电流检测技术复杂，其新药筛选模型极其缺乏。因此，本研究利用非洲爪蟾卵母细胞表达系统，进行CaV2。2体外重组表达，建立电生理学技术药物筛选模型，并对该表达技术体系进行了优化(本研究获得广西大学伦理委员会审查批准，批准号:GXU-2023-0249)。首先，以大鼠CaV2。2的主亚基a1B及其辅助亚基a2δ1和β3的cDNA基因为模板，分别在体外进行转录，人工合成了CaV2。2 3个亚基的mRNA(cRNA)，按照2∶1∶1的质量比混合，将3种cRNA注射到非洲爪蟾卵母细胞中进行表达。随后使用双电极电压钳技术检测CaV2。2离子通道是否产生细胞膜内向电流，同时对各个表达条件进行了优化，从通道的激活、失活等方面表征了其门控功能。结果显示，在cRNA显微注射后的第3～5天，使用四乙基氢氧化铵(TEAOH)和1，2-双(2-氨基苯氧基)乙烷-N，N，N'，N'-四乙酸四酯(BAPTA-AM)处理，可分别消除内源性钾离子通道和Ca2+激活的氯离子通道干扰，成功检测到稳定的CaV2。2介导的钡离子电流。CaV2。2的最大激活膜电位为0 mV，当膜电位大于+50 mV时会出现电流方向反转。通过拟合稳态激活和失活曲线获知CaV2。2的半激活电位和半失活电位分别为-15。9和-60。2 mV。本研究基于非洲爪蟾卵母细胞，通过条件优化，建立了稳定的CaV2。2表达系统。该体外表达系统可以为靶向CaV2。2活性化合物或先导药物的筛选提供新的途径。

外文标题：Establishment and optimization of drug screening model for N-type voltage-gated calcium channels in Xenopus laevis oocyte expression system

外文摘要：N-type voltage-gated calcium(Ca2+)channels(N-type VGCC,CaV2.2)mediate Ca2+influx in response to action potential at the presynaptic terminal,and play an important role in synaptogenesis,neurotransmitter release and nociceptive signal transduction.It is a new target for the development of drugs for the treatment of neuralgia(chronic pain)and other major diseases.Due to the difficulty of calcium channel expression in vitro and the detection of channel current,there is a great lack of new drug screening models.In this study,we established and optimized the electrophysiological drug screening model using Xenopus laevis oocytes for the recombinant expression of CaV2.2 in vitro(this study were reviewed and approved by the Ethics Committee of Guangxi University,approval number:GXU-2023-0249).Firstly,the linear plasmids encoding cDNA of major subunit α1B and auxiliary subunits α2δ1 and β3 of rat CaV2.2 were used as templates for in vitro transcription to generate their related mRNA(cRNA),after which three kinds of cRNA were injected into Xenopus laevis oocytes at the mass ratio of 2∶1∶1 for expression.The two-electrode voltage clamp(TEVC)technique was used to detect the inward current produced by CaV2.2.At the same time,the expression conditions of CaV2.2 were optimized,and its gating function was characterized from the aspects of channel activation and inactivation.The results showed that 3-5 days after cRNA microinjection,stable CaV2.2-mediated barium ion(Ba2+)currents were successfully detected.The interference of endogenous potassium channels and Ca2+-activated chloride channels can be eliminated by tetraethylammonium hydroxide(TEAOH)and 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(BAPTA-AM)treatment.The maximum potential for CaV2.2 activation is 0 mV,and the current reverses to be outward when the membrane potential is greater than+50 mV.By fitting the steady-state activation and inactivation curves,the half-maximal activation potential and half-maximal inactivation potential of CaV2.2 are identified as-15.9 and-60.2 mV.In this study,a stable CaV2.2 expression system was established based on Xenopus laevis oocytes.The in vitro expression system can provide a new way for the screening of CaV2.2 active compounds or lead drugs.

外文关键词：

voltage-gated calcium channeltwo-electrode voltage clampXenopus laevis oocyteelectrophysiologygating characteristics

作者：

秦源、崔城、朱晓鹏、长孙东亭、于津鹏、罗素兰

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作者单位：

广西大学医学院,广西特色生物医药重点实验室,广西南宁 530004

关键词：

电压门控钙离子通道双电极电压钳非洲爪蟾卵母细胞电生理门控特性

基金：

国家重点研发计划项目广西自然科学基金资助项目国家自然科学基金资助项目广西科技基地和人才专项

项目编号：

2022YFE01327002022GXNSFBA03566282104059桂科AD22035948

出版年：

2024

DOI：

10.16438/j.0513-4870.2024-0156

药学学报

中国药学会中国医学科学院药物研究所

药学学报

CSTPCD北大核心

影响因子：1.274

ISSN：0513-4870

年,卷(期)：2024.59(7)

参考文献量44