α6/α3β4烟碱型乙酰胆碱受体β4亚基双点突变体的制备及其功能研究  

Preparation and Functional Study of β4 Subunit Double-point Mutant of α6/α3β4 Nicotinic Acetylcholine Receptors

作　　者：李旭扬 陈舒苗 于津鹏 罗素兰[1] 长孙东亭[1] LI Xuyang;CHEN Shumiao;YU Jinpeng;LUO Sulan;ZHANGSUN Dongting(School of Life and Pharmaceutical Sciences/Ministry of Education Key Laboratory of Tropical Biological Resources/Haikou Key Laboratory for Marine Drugs,Hainan University,Haikou,Hainan 570228;Medical College,Guangxi University,Nanning,Guangxi 530004,China)

机构地区：[1]海南大学热带生物资源教育部重点实验室/海口市海洋药物重点实验室/生命科学与药学院,海口570228 [2]广西大学医学院,南宁530004

出　　处：《热带生物学报》2021年第4期403-411,共9页Journal of Tropical Biology

基　　金：国家自然科学基金项目(41966003)。

摘　　要：在烟碱型乙酰胆碱受体家族中,含α6亚型的受体是比较特殊的一类。研究发现α6β4^(*)亚型(^(*)表示含其他亚基)在神经系统方面具有一定的调节作用,与神经性疼痛、成瘾等多种疾病的发病机制密切相关,可作为潜在的治疗靶点。比对大鼠与人类α6和β4亚基的序列,以野生型基因为模板,采用基因定点突变的方式,成功得到3种相邻位点突变的α6/α3β4受体突变型基因。通过体外转录、显微注射等手段,构建α6/α3β4受体野生型和突变型在非洲爪蟾卵母细胞上的表达模型,并使用双电极电压钳检测受体的表达情况。结果表明,与野生型受体相比,突变体α6/α3β4[S52N, I53V]对ACh的敏感性有所提升,EC_(50)为59.58μmol·L^(-1),是野生型的1/2,而其他两种突变体的敏感性与野生型相同。The receptor containing α6 subunit is one of the special nicotinic acetylcholine receptors. Recent studies have found that the α6/α3β4 subtypes have a certain regulatory effect on the nervous system. And it is related to the pathogenesis of many diseases, such as neuropathic pain, addiction, etc., which have been regarded as potential therapeutic targets. The sequences of rat and human α6 and β4 subunits were compared,and three mutant α6/α3β4 receptor genes with adjacent site mutations were produced on the basis of wild-type genes by site-directed mutagenesis. The α6/α3β4 receptors and their mutants were expressed on Xenopus oocytes by transcription in vitro, microinjection and other methods. And the functions of the receptors were detected by using the two-electrode voltage voltage-clamp system. The results indicated that the mutant receptor α6/α3β4 [S52 N, I53 V] was more sensitive to ACh, with its EC_(50)being 59.58 μmol·L^(-1), which is half of the wild-type receptor. Meanwhile, the other two mutant receptors had similar sensitivities to the wild type.

关 键 词：α6/α3β4烟碱型乙酰胆碱受体 相邻位点突变 体外表达 双电极电压钳 

分 类 号：Q78[生物学—分子生物学] R91[医药卫生—药学]