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Uncovering the lipidic basis for the preparation of functional nicotinic acetylcholine receptor detergent complexes for structural studies.

ABSTRACT: This study compares the lipid composition, including individual phospholipid molecular species of solubilized nAChR detergent complexes (nAChR-DCs) with those of the bulk lipids from their source, Torpedo californica (Tc) electric tissue. This lipidomic analysis revealed seventy-seven (77) phospholipid species in the Tc tissue. Analysis of affinity-purified nAChR-DCs prepared with C-12 to C-16 phospholipid analog detergents alkylphosphocholine (FC) and lysofoscholine (LFC) demonstrated that nAChR-DCs prepared with FC12, LFC14, and LFC16 contained >60 phospholipids/nAChR, which was more than twice of those prepared with FC14, FC16, and LFC12. Significantly, all the nAChR-DCs lacked ethanolamine and anionic phospholipids, contained only four cholesterol molecules, and a limited number of phospholipid molecular species per nAChR. Upon incorporation into oocytes, FC12 produce significant functionality, whereas LFC14 and LFC16 nAChR-DCs displayed an increased functionality as compared to the crude Tc membrane. All three nAChR-DCs displayed different degrees of alterations in macroscopic activation and desensitization kinetics.

SUBMITTER: Quesada O

PROVIDER: S-EPMC5027579 | biostudies-literature | 2016 Sep

REPOSITORIES: biostudies-literature

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Uncovering the lipidic basis for the preparation of functional nicotinic acetylcholine receptor detergent complexes for structural studies.

Quesada Orestes O González-Freire Carol C Ferrer María Carla MC Colón-Sáez José O JO Fernández-García Emily E Mercado Juan J Dávila Alejandro A Morales Reginald R Lasalde-Dominicci José A JA

Scientific reports 20160919

This study compares the lipid composition, including individual phospholipid molecular species of solubilized nAChR detergent complexes (nAChR-DCs) with those of the bulk lipids from their source, Torpedo californica (Tc) electric tissue. This lipidomic analysis revealed seventy-seven (77) phospholipid species in the Tc tissue. Analysis of affinity-purified nAChR-DCs prepared with C-12 to C-16 phospholipid analog detergents alkylphosphocholine (FC) and lysofoscholine (LFC) demonstrated that nACh ...[more]

PMID: 27641515

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Project description:For a long time, traditional purification and extraction methods for the native Torpedo californica nicotinic acetylcholine receptor in lipid-like detergent complex (nAChR-DC) have compromised its purity, functionality and X-ray structural studies possibility. The dataset presented in this article provide a characterization of the Torpedo californica nAChR-DC purified using a sequential purification processes developed in our laboratory [1]. This purification takes in consideration all of the physicochemical and functional requirements stablished by several researchers for the past three decades for the nAChR. These requirements were addressed in order to preserve the stability and functionality of nAChR-DC while ensuring the highest degree of protein purity. We focused on the effect of cholesteryl hemisuccinate (CHS) supplementation on nAChR conformational changes during the purification process. Data from the size exclusion chromatography of the nAChR-DC supplemented with CHS in concentrations ranging from 0.01 mM, 0.1 mM, 0.2 mM and 0.5 mM consistently demonstrated that 0.5 mM CHS affects receptor stability via disassemble of the pentameric oligomer. However, 0.2 mM CHS produced negligible nAChR-DC subunit disruption. The purified nAChR-DC has been characterized by circular dichroism (CD) and fluorescence recovery after photobleaching (FRAP), in order to assess its stability. The CD data was recorded in the wavelength range of 190-250 nm, showed that CHS induce a ⍺-helix to β-sheet transition of the nAChR-DC. The nAChR-LFC-16 delipidation with Methyl-β-Cyclodextrin decreased the percentage of α-helix and increased the β-sheet antiparallel secondary structure and levels the percentage of turns to that of the nAChR-DC without CHS treatment. Additionally, the stability of the nAChR-DC supplemented with CHS and incorporated into lipid cubic phase (LCP) was monitored for a period of 30 days by means of FRAP. The LCP-FRAP data allowed to establish possible optimal crystallization conditions for the development of crystals from purified nAChR-conjugated to α-Bungarotoxin, Alexa Fluor ™ 488 (α-BTX) in order to obtain a high-resolution atomic structure by X-ray diffraction.

Project description:The nicotinic acetylcholine receptor (nAChR) of Torpedo electric rays has been extensively characterized over the last three decades. However, high-resolution structural studies have been hampered by the lack of mechanistic molecular models that describe how detergents influence membrane protein stability and function. Furthermore, elucidation of the dynamic detergent-lipid-protein interactions of solubilized membrane proteins is a largely unexplored research field. This study examines the effects of nine detergents on: (1) nAChR-lipid composition (gas chromatography with flame ionization; GC-FID and/or mass selective detectors; GC-MSD), (2) stability and aggregation state (analytical size exclusion chromatography; A-SEC and electron microscopy; EM) and (3) ion channel function (planar lipid bilayers). Detergent solubilization of nAChR-enriched membranes did not result in significant native lipid depletion or destabilization. Upon purification, native lipid depletion occurred in all detergents, with lipid-analogue detergents CHAPS {(3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate}, FC-12 (n-dodecylphosphocholine) and sodium cholate (3alpha,7alpha,12alpha-trihydroxy-5beta-cholan-24-oic acid) maintaining stability and supporting ion channel function, and non-lipid-analogue detergents Cymal-6 (6-cyclohexyl-1-hexyl-beta-D-maltoside), DDM (n-dodecyl-beta-D-maltopyranoside), LDAO (lauryldimethylamine-N-oxide) and OG (n-octyl-beta-d-glucopyranoside) decreasing stability and significantly reducing or completely suppressing ion channel function. Anapoe-C(12)E(9 )(polyoxyethylene-[9]-dodecyl ether) and BigCHAP (N,N'-bis-[3-d-gluconamidopropyl] cholamide) retained residual amounts of native lipid, maintaining moderate stability and ion channel function compared to lipid-analogue detergents. Therefore, the nAChR can be stable and functional in lipid-analogue detergents or in detergents that retain moderate amounts of residual native lipids, but not in non-lipid-analogue detergents.

Dataset Information

Uncovering the lipidic basis for the preparation of functional nicotinic acetylcholine receptor detergent complexes for structural studies.

Publications

Uncovering the lipidic basis for the preparation of functional nicotinic acetylcholine receptor detergent complexes for structural studies.

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