Assessment of Purity, Functionality, Stability, and Lipid Composition of Cyclofos-nAChR-Detergent Complexes from Torpedo californica Using Lipid Matrix and Macroscopic Electrophysiology.

IF 2.3 4区生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY

Journal of Membrane Biology Pub Date : 2023-06-01 DOI:10.1007/s00232-023-00285-x

Orestes Quesada, Joel E González-Nieves, José Colón, Rafael Maldonado-Hernández, Carol González-Freire, Jesús Acevedo-Cintrón, Irvin D Rosado-Millán, José A Lasalde-Dominicci

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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.

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用脂质基质和宏观电生理学评价加利福尼亚鱼雷中cyclofos - nachr -洗涤剂复合物的纯度、功能、稳定性和脂质组成。

本研究的主要目的是寻找能够维持加利福尼亚鱼雷烟碱乙酰胆碱受体(Tc-nAChR)功能和稳定性的洗涤剂。我们检测了亲和纯化的Tc-nAChR在Cyclofos (CF)家族洗涤剂中的功能、稳定性和纯度分析[cyclofoscholine 4 (CF-4)、cyclofoscholine 6 (CF-6)和cyclofoscholine 7 (CF-7)]。采用双电极电压钳法(TEVC)评价cf - tc - nachr -洗涤剂复合物(DC)的功能。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)。我们还使用超高效液相色谱(UPLC)和电喷雾电离质谱(ESI-MS/MS)进行了脂质组学分析，以评估CF-Tc-nAChR-DCs的脂质组成。CF-4-Tc-nAChR-DC具有较强的宏观电流(- 200±60 nA);而CF-6-Tc-nAChR-DC和CF-7-Tc-nAChR-DC的宏观电流明显降低。CF-6-Tc-nAChR和CF-4-Tc-nAChR表现出更高的分数荧光恢复。胆固醇的加入对CF-6-Tc-nAChR的移动部分产生了轻微的增强。脂质组学分析显示，CF-7-Tc-nAChR-DC显示出大量的脱脂，这与该复合物缺乏稳定性和功能反应一致。虽然CF-6-nAChR-DC复合物保留了最多的脂质，但它显示出6种脂质的损失[SM(d16:1/18:0);电脑(18:2/14:1);电脑(14:0/18:1);电脑(16:0/18:1);PC(20:5/20:4)和PC(20:4/20:5)]存在于CF-4-nAChR-DC中。总体而言，CF-4- nachr表现出强大的功能，显著的稳定性和三种CF洗涤剂中最好的纯度;因此，CF-4是制备Tc-nAChR晶体进行结构研究的合适候选材料。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Journal of Membrane Biology 生物-生化与分子生物学

CiteScore

4.80

自引率

4.20%

发文量

审稿时长

6-12 weeks

期刊介绍： The Journal of Membrane Biology is dedicated to publishing high-quality science related to membrane biology, biochemistry and biophysics. In particular, we welcome work that uses modern experimental or computational methods including but not limited to those with microscopy, diffraction, NMR, computer simulations, or biochemistry aimed at membrane associated or membrane embedded proteins or model membrane systems. These methods might be applied to study topics like membrane protein structure and function, membrane mediated or controlled signaling mechanisms, cell-cell communication via gap junctions, the behavior of proteins and lipids based on monolayer or bilayer systems, or genetic and regulatory mechanisms controlling membrane function. Research articles, short communications and reviews are all welcome. We also encourage authors to consider publishing ''negative'' results where experiments or simulations were well performed, but resulted in unusual or unexpected outcomes without obvious explanations. While we welcome connections to clinical studies, submissions that are primarily clinical in nature or that fail to make connections to the basic science issues of membrane structure, chemistry and function, are not appropriate for the journal. In a similar way, studies that are primarily descriptive and narratives of assays in a clinical or population study are best published in other journals. If you are not certain, it is entirely appropriate to write to us to inquire if your study is a good fit for the journal.