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Abstract：
The main objective of the present study was to find detergents that can maintain the functionality and stability of the Torpedo californica nicotinic acetylcholine receptor (Tc-nAChR). We examined the functionality, stability, and purity analysis of affinity-purified Tc-nAChR solubilized in detergents from the Cyclofos (CF) family [cyclofoscholine 4 (CF-4), cyclofoscholine 6 (CF-6), and cyclofloscholine 7 (CF-7)]. The functionality of the CF-Tc-nAChR-detergent complex (DC) was evaluated using the Two Electrode Voltage Clamp (TEVC) method. To assess stability, we used the florescence recovery after photobleaching (FRAP) in Lipidic Cubic Phase (LCP) methodology. We also performed a lipidomic analysis using Ultra-Performance Liquid Chromatography (UPLC) coupled to electrospray ionization mass spectrometry (ESI-MS/MS) to evaluate the lipid composition of the CF-Tc-nAChR-DCs. The CF-4-Tc-nAChR-DC displayed a robust macroscopic current (- 200 ± 60 nA); however, the CF-6-Tc-nAChR-DC and CF-7-Tc-nAChR-DC displayed significant reductions in the macroscopic currents. The CF-6-Tc-nAChR and CF-4-Tc-nAChR displayed higher fractional florescence recovery. Addition of cholesterol produced a mild enhancement of the mobile fraction on the CF-6-Tc-nAChR. The lipidomic analysis revealed that the CF-7-Tc-nAChR-DC displayed substantial delipidation, consistent with the lack of stability and functional response of this complex. Although the CF-6-nAChR-DC complex retained the largest amount of lipids, it showed a loss of six lipid species [SM(d16:1/18:0); PC(18:2/14:1); PC(14:0/18:1); PC(16:0/18:1); PC(20:5/20:4), and PC(20:4/20:5)] that are present in the CF-4-nAChR-DC. Overall, the CF-4-nAChR displayed robust functionality, significant stability, and the best purity among the three CF detergents; therefore, CF-4 is a suitable candidate to prepare Tc-nAChR crystals for structural studies.
摘要：
本研究的主要目的是找到可以维持加利福尼亚鱼雷烟碱乙酰胆碱受体（Tc-nAChR）的功能和稳定性的洗涤剂。我们检查了功能，稳定性,和纯度分析在Cyclofos（CF）家族的洗涤剂中溶解的亲和纯化的Tc-nAChR[cyclofocolinine4（CF-4），环福林6(CF-6)，和环弗罗林7号(CF-7)]。使用双电极电压钳(TEVC)方法评估CF-Tc-nAChR-洗涤剂复合物(DC)的功能性。为了评估稳定性，我们在脂质立方相（LCP）方法中使用了光漂白后的荧光恢复（FRAP）。我们还使用与电喷雾电离质谱(ESI-MS/MS)偶联的超高效液相色谱(UPLC)进行脂质组学分析,以评估CF-Tc-nAChR-DC的脂质组成。CF-4-Tc-nAChR-DC显示出强大的宏观电流（-200±60nA）；但是，CF-6-Tc-nAChR-DC和CF-7-Tc-nAChR-DC显示宏观电流显着降低。CF-6-Tc-nAChR和CF-4-Tc-nAChR显示出较高的荧光恢复分数。胆固醇的添加使CF-6-Tc-nAChR上的移动部分略有增强。脂质组学分析显示，CF-7-Tc-nAChR-DC表现出实质性的脱脂作用，与该复合物缺乏稳定性和功能响应相一致。尽管CF-6-nAChR-DC复合物保留了最大量的脂质，它显示了六个脂质种类的损失[SM（d16：1/18：0）;PC（18：2/14：1）;PC（14：0/18：1）;PC（16：0/18：1）;PC（20：5/20：4），和PC(20:4/20:5)]存在于CF-4-nAChR-DC中。总的来说,CF-4-nAChR显示出强大的功能，显著的稳定性，三种CF洗涤剂中纯度最好；因此，CF-4是制备Tc-nAChR晶体用于结构研究的合适候选物。
