用电压钳检测烟碱型乙酰胆碱受体导电孔道的重要位点  

Identifying of important sites of the conducting pore of nicotinic acetycholine receptor through two-electrode voltage-clamp

作　　者：薛奋勤 李华 刘丽娜 许晴 薛冰 魏华 XUE Fen-qin;LI Hua;LIU Li-na;XU Qing;XUE Bing;WEI Hua(Core Facility Center of Capital Medical University,Beijing 100069,China)

机构地区：[1]首都医科大学中心实验室,北京100069

出　　处：《基础医学与临床》2021年第9期1309-1312,共4页Basic and Clinical Medicine

基　　金：首都医科大学校长基金技术类(2014JS18)。

摘　　要：目的利用双电极电压钳技术检测组成人烟碱型乙酰胆碱受体离子导电孔道的主要氨基酸位点。方法用定点突变的方法将人alpha 7烟碱型乙酰胆碱受体(α7 nAChR)亚基跨膜区段M2的氨基酸(Leu248–Ala258)逐一突变为亲水性的丝氨酸,随后利用cDNA质粒体外转录获得cRNA,并将单个突变体的cRNA注射到非洲爪蟾卵母细胞中。注射1~3 d后,利用双电极电压钳检测野生型(WT)α7 nAChR及各突变体的功能。结果获得跨膜区段M2亲水性突变后,与WT相比,发现4个亲水性突变体(L248、V252、L255和L256)对激动剂乙酰胆碱(ACh)的EC 50值明显降低(P<0.001);剂量-反应曲线向左显著移动,脱敏程度明显减轻(P<0.01);而且它们对ACh的敏感性与脱敏程度呈正相关性(R 2=0.98)。结论α7 nAChR亚基的M2跨膜区段的4个位点L248、V252、L256和L255是组成其导电孔道的主要氨基酸位点。Objective To detect the main amino acid sites in the ion-conducting pore of the humanα7 nicotinic acetycholine receptor(α7nAChR)by two-electrode voltage-clamp.Methods The amino acids in the transmembrane region M2 ofα7 nAChR subunit(from Leu248 to Ala258)were mutated to hydrophilic serine by site-directed mutagenesis.cRNA was synthesized in vitro and injected into Xenopus oocytes.After 1~3 days,the function of wild typeα7 nAChR and its mutants were detected by two-electrode voltage-clamp.Results After the hydrophilic mutation on the second transmembrane domain,it was indicated that the mutations of the hydrophobic residues(L248,V252,L255 and L256)to hydrophilic ones significantly decrease the EC 50 values of the receptor to agonist acetylcholine.The hydrophilic mutations of these four M2 residues resulted in a dramatic leftward shift of the dose-response curve and decrease desensitization.Moreover,the desensitization rate was positively correlated to the EC 50 values in log scale.Conclusions The residues L248,V252,L256 and L255 in the transmembrane M2 ofα7 nAChR are the main sites to construct the ion-conducting pore.

关 键 词：α7烟碱受体 双电极电压钳 离子导电孔道 

分 类 号：Q789[生物学—分子生物学] R34[医药卫生—基础医学]