烟碱型乙酰胆碱受体α3[K152E,E184D,Q195T]β4突变型的构建及其功能研究

Construction and functional evaluation of an α3[K152E,E184D,Q195T]β4 nicotinic acetylcholine receptor mutant

α3β4烟碱型乙酰胆碱受体(nicotinic acetylcholine receptors,nAChRs)是成瘾、癌症和肥胖等重要疾病的潜在新靶点。本研究对大鼠(rat,r)α3β4 nAChRs的α3亚基上的3个氨基酸位点同时进行突变,将这3个位点分别突变为rα6亚基上与α3亚基上相对应的氨基酸种类,构建α3[K152E,E184D,Q195T]β4三点突变型受体,并研究其功能。利用PCR介导的定点突变方法构建了α3[K152E,E184D,Q195T]三点突变体载体,体外转录获得相应的cRNA,与野生型β4亚基的cRNA按相同比例注射到非洲爪蟾卵母细胞中进行重组表达,然后用双电极电压钳技术检测其受体活性和功能。测定α3β4 nAChRs野生型和突变型在乙酰胆碱、尼古丁和金雀花碱3种不同激动剂的诱导下,其配体门控电流的大小以及门控特征,比较野生型和突变型受体之间的功能差异。α3[K152E,E184D,Q195T]三点突变体的功能与野生型相比存在显著差异。乙酰胆碱、尼古丁和金雀花碱对α3β4 nAChR野生型的半数最大效应浓度(EC_(50))分别为277.5、34.02和23.05μmol·L^(-1);针对三点突变体,3种激动剂的EC_(50)分别为170.5、26.6和98.45μmol·L^(-1)。3种激动剂对突变体的EC_(50)与野生型受体的EC_(50)相比,其活性变化分别为0.6、0.8和4.3倍。其中突变型受体对金雀花碱的活性影响最显著,其激动剂活性下降了77%。此外,与1 mmol·L^(-1)乙酰胆碱诱导的峰值电流幅度相比,金雀花碱对野生型和突变型α3β4 nAChRs的最大激动效率(E_(max))从94.12%提升至155.08%。α3[K152E,E184D,Q195T]β4三点突变型明显降低了对金雀花碱的敏感性,但其最大激动电流幅度明显变大。三点突变型略微增强了对乙酰胆碱和尼古丁的敏感性,说明α3亚基上的这3个氨基酸对α3β4 nAChRs的配体结合功能影响较大,对不同激动剂的影响情况各异,这为今后探究α3β4 nAChRs重要受体的精细结构和功能以及相关疾病的发病机制研究提供了很好的线索。

α3β4 nicotinic acetylcholine receptors(nAChRs)are potential therapeutic targets in diseases such as addiction,cancer,and obesity.In this study,by replacing three amino acids of theα3 subunit with the corresponding positions of the rα6 subunit simultaneously,anα3[K152E,E184D,Q195T]subunit mutant was constructed by PCR-mediated site-directed mutagenesis and its cRNA was obtained by in vitro transcription.The cRNA of mutant subunits mixed in equal molar ratios withβ4 subunits were microinjected into Xenopus oocytes.The pharmacological activity and function ofα3[K152E,E184D,Q195T]β4 nAChR was evaluated by a two-electrode voltage clamp electrophysiological technique.Acetylcholine,nicotine,and cytisine were used as agonists to evaluate the magnitude of ligand-gated currents and gating characteristics of wild-type and mutantα3β4 nAChRs.The half-maximal effective concentrations(EC_(50))of acetylcholine,nicotine,and cytisine on wild-typeα3β4 nAChRs were 277.5,34.02 and 23.05µmol·L^(-1),respectively,while their EC_(50) values withα3[K152E,E184D,Q195T]β4 nAChR were 170.5,26.6,and 98.45µmol·L^(-1),respectively.Thus these EC_(50) values for the three agonists towards the mutant receptor were changed 0.6-fold,0.8-fold,and 4.3-fold,respectively,compared with the wild-type receptor;cytisine was most strongly affected,with a 77%decrease in potency.However,the maximum agonistic efficiency(E_(max))of cytisine on wild-type and mutantα3β4 nAChRs was increased from 94.12%to 155.08%relative to the peak current amplitude induced by 1 mmol·L^(-1)acetylcholine.Thus,although theα3[K152E,E184D,Q195T]β4 nAChR had significantly reduced sensitivity to cytisine,the maximum current amplitude induced by cytisine was clearly increased.This mutant had slightly increased sensitivity to acetylcholine and nicotine.The results indicate that these three amino acids of theα3 subunit have important and varying effects on ligand binding to theα3β4 nAChR,providing a basis for further structure-functional research onα3β4 nAChR,as well as the pathology of related diseases.