α7烟碱型乙酰胆碱受体分子伴侣Tmem35a的克隆及其功能研究  

Cloning and functional characterization ofα7 nicotinic acetylcholine receptor molecular chaperone Tmem35a

作　　者：王紫涵 于津鹏 长孙东亭 朱晓鹏 罗素兰 WANG Zi-han;YU Jin-peng;ZHANGSUN Dong-ting;ZHU Xiao-peng;LUO Su-lan(Guangxi Key Laboratory of Special Biomedicine,School of Medicine,Guangxi University,Nanning 530004,China)

机构地区：[1]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

出　　处：《药学学报》2024年第7期1993-2001,共9页Acta Pharmaceutica Sinica

基　　金：国家重点研发计划政府间国际科技创新合作重点专项(2022YFE0132700);广西科技基地和人才专项(桂科AD22035948);国家自然科学基金资助项目(42376112,82320108019,82360698)。

摘　　要：烟碱型乙酰胆碱受体(nicotinic acetylcholine receptors,nAChRs)属于配体门控离子通道受体,其中α7nAChR亚型广泛分布于大脑皮层、丘脑和海马体等区域。此外,在小胶质细胞、巨噬细胞、骨髓细胞等也有分布,研究发现其与胆碱能抗炎通路的功能密切相关,是阿尔茨海默症和精神分裂症药物开发的重要靶标。建立稳定的体外药物筛选体系,对于靶向α7 nAChR新药的高效筛选至关重要。在非洲爪蟾卵母细胞膜上,重组表达nAChRs的不同亚型,并通过电生理技术进行电流检测,是一种先进而复杂的新药筛选模型。分子伴侣可以协助部分nAChRs亚基组装形成功能性受体,为靶向该受体的化合物筛选提供稳定表达的模型。为此,本研究从大鼠体内分离克隆了α7 nAChR的一个分子伴侣基因,其名称为Tmem35a(transmembrane protein 35A),进一步构建了该基因的重组表达载体,再利用体外转录技术获得了该基因的cRNA,将之与α7 nAChR的cRNA混合后同时注射到非洲爪蟾卵母细胞中进行表达。然后,利用双电极电压钳检测该分子伴侣对α7 nAChR电流表达和通道药理活性的影响。结果显示,TMEM35A(也被称为novel acetylcholine receptor chaperone,NACHO)可有效提高α7 nAChR蛋白在卵母细胞膜上的表达,受体蛋白表达量提高了约1倍;其配体乙酰胆碱(acetylcholine,ACh)刺激诱发的峰值电流提高了约10倍。注入Tmem35a cRNA后的卵母细胞,其表达的α7 nAChR对激动剂ACh的半数效应浓度为228.5μmol·L^(-1),和本体223.3μmol·L^(-1)基本一致,维持了α7受体正常功能特性。研究结果表明,分子伴侣NACHO有效协助了α7 nAChR在非洲爪蟾卵母细胞的异源表达,稳定表达的α7 nAChR可以为靶向该受体的先导化合物活性筛选提供模型。本研究所有动物实验过程经广西大学伦理委员会审查批准(批准号:GXU-2023-0249)。Nicotinic acetylcholine receptors(nAChRs)belong to ligand-gated ion channel receptors,of whichα7 nAChR subtype is widely distributed in the cerebral cortex,thalamus,hippocampus,and also identified in microglia,macrophages,bone marrow cells,etc.Previous studies revealed thatα7 nAChR is closely related to the function of the cholinergic anti-inflammatory pathway,and is a vital target for drug development of Alzheimer's disease and schizophrenia.The establishment of a stableα7 nAChR in vitro drug screening system is crucial for the efficient screening of novel drugs targeting this target.Recombinant expression of different subtypes of nAChRs on Xenopus laevis oocyte membranes and current detected by two-electrode voltage clamp(TEVC)is an advanced and complex model for novel drug screening.Molecular chaperones can assist the assembly of some nAChR subunits to form functional receptors,providing a stable expression model for the screening of compounds targeting this receptor.In this study,a molecular chaperone gene ofα7 nAChR,transmembrane protein 35A(Tmem35a),was isolated and cloned from rats.We constructed the recombinant expression vector and obtained the cRNA of Tmem35a by in vitro transcription technique.Two cRNAs(Tmem35a andα7)were mixed and injected into X.laevis oocytes for expression.Then,the effects of this molecular chaperone on the current expression and pharmacological properties ofα7 nAChR were evaluated by the TEVC.The results revealed that TMEM35A,also known as novel acetylcholine receptor chaperone(NACHO)could effectively increase the expression ofα7 nAChR protein on oocyte membranes,and the amount ofα7 nAChR protein was increased about 1-fold.The peak current induced by agonist acetylcholine(ACh)was increased about 10-fold.After injection of Tmem35a cRNA,the median effect concentration(EC_(50))value ofα7 nAChR to agonist ACh is 228.5μmol·L^(-1),which shows almost no difference from nativeα7 nAChR(EC_(50):223.3μmol·L^(-1)),indicating the preservation of the normal properties ofα7 nAChR.

关 键 词：Α7烟碱型乙酰胆碱受体 分子伴侣NACHO 基因克隆 双电极电压钳 药物筛选模型 

分 类 号：R966[医药卫生—药理学]