【摘要】 目的:探讨乙酰胆碱、尼古丁、伐尼克兰和金雀花碱4种激动剂与α4β2烟碱型乙酰胆碱受体（nAChRs）及其突变体相互作用的活性关键位点。方法:体外转录法制备野生型（wt）与突变型α4亚基cRNAs,注射入非洲爪蟾卵母细胞,应用电生理方法,测定4种激动剂对α4β2 nAChRs及其突变体的门控活性。结果:乙酰胆碱对wt α4β2、α4（R188I）β2、α4（P198Q）β2和α4（R188I,P198Q）β2 nAChRs的半数有效浓度(EC₅₀)分别为67.15、63.83、22.43和1.37μmol/L,尼古丁对wt α4β2、α4（R188I）β2、α4（P198Q）β2和α4（R188I,P198Q）β2nAChRs的EC₅₀分别为1.43、0.63、1.05和0.27μmol/L。与乙酰胆碱（10 mmol/L）诱导的峰值电流幅度相比,金雀花碱对wt α4β2和α4（R188I,P198Q）β2 nAChRs的最大激动效率(E_max)从37.62%提升至95.14%,伐尼克兰对wt α4β2和α4（R188I,P198Q）β2 nAChRs的E_max也从67.94%提升至94.69%。结论:α4亚基第188位精氨酸（arginine,Arg,R）和第198位脯氨酸（proline,Pro,P）分别突变成异亮氨酸（isoleucine,Ile,I）和谷氨酰胺（glutamine,Gln,Q）后,增强了α4β2 nAChRs对乙酰胆碱和尼古丁的敏感性,并提高了α4β2 nAChRs对金雀花碱与伐尼克兰的通道开放效率。更多还原

【Abstract】 AIM:To explore the key sites of interaction between 4 different agonists（acetylcholine,nicotine,varenicline and cytisine）and α4β2 nicotinic acetylcholine receptors（nAChRs）.METHODS:The cRNAs of wild-type（wt）and mutant α4 subunits were synthesized by in vitro transcription using SP6 RNA polymerase. The cRNAs of α4β2 nAChRs were microinjected into Xenopus oocytes. Gating properties for wt and mutant α4β2 nAChRs were detected by two-electrode voltage clamp technique using the 4 agonists.RESULTS:The acetylcholine concentration of 50% maximal effect(EC₅₀)for wt α4β2,α4（R188 I）β2,α4（P198 Q）β2 and α4（R188 I,P198 Q）β2 nAChRs was 67. 15,63. 83,22. 43 and 1. 37 μmol/L,respectively. The EC₅₀ of nicotine for wt α4β2,α4（R188 I）β2,α4（P198 Q）β2 and α4（R188 I,P198 Q）β2 nAChRs was 1. 43,0. 63,1. 05 and 0. 27 μmol/L,respectively. Compared with the peak current amplitude induced by acetylcholine（10 mmol/L）,the maximum efficacy(E_max)of cytisine for wt α4β2 and α4（R188 I,P198 Q）β2 nAChRs was increased from 37. 62% to 95. 14%. The E_max of varenicline for wt α4β2 and α4（R188 I,P198 Q）β2 nAChRs was increased from 67. 94% to 94. 69%.CONCLUSION:The sensitivity of α4β2 nAChRs to acetylcholine and nicotine was enhanced when the arginine（Arg,R）-188 and proline（Pro,P）-198 in the α4 subunit were mutated to isoleucine（Ile,I）and glutamine（Gln,Q）,respectively. Meanwhile,the channel opening efficiency of α4β2 nAChRs to cytisine and varenicline was also improved significantly.更多还原