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Crystallization scale purification of ?7 nicotinic acetylcholine receptor from mammalian cells using a BacMam expression system.

ABSTRACT: To report our methods for expression and purification of ?7 nicotinic acetylcholine receptor (?7-nAChR), a ligand-gated pentameric ion channel and an important drug target.?7-nAChRs of 10 different species were cloned into an inducible BacMam vector with an N-terminal tag of a tandem maltose-binding protein (MBP) and a TEV cleavage site. This ?7-nAChR fusion receptor was expressed in mammalian HEK293F cells and detected by Western blot. The expression was scaled up to liters. The receptor was purified using amylose resin and size-exclusion chromatography. The quality of the purified receptor was assessed using SDS-PAGE gels, thermal stability analysis, and negative stain electron microscopy (EM). The expression construct was optimized through terminal truncations and site-directed mutagenesis.Expression screening revealed that ?7-nAChR from Taeniopygia guttata had the highest expression levels. The fusion receptor was expressed mostly on the cell surface, and it could be efficiently purified using one-step amylose affinity chromatography. One to two milligrams of the optimized ?7-nAChR expression construct were purified from one liter of cell culture. The purified ?7-nAChR samples displayed high thermal stability with a Tm of 60 °C, which was further enhanced by antagonist binding but decreased in the presence of agonist. EM analysis revealed ring-like structures with a central hydrophilic hole, which was consistent with the pentameric assembly of the ?7-nAChR channel.We have established methods for crystallization scale expression and purification of ?7-nAChR, which lays a foundation for high-resolution structural studies using X-ray crystallography or single particle cryo-EM analysis.

SUBMITTER: Cheng H

PROVIDER: S-EPMC4564878 | biostudies-literature | 2015 Aug

REPOSITORIES: biostudies-literature

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Crystallization scale purification of α7 nicotinic acetylcholine receptor from mammalian cells using a BacMam expression system.

Cheng Hao H Fan Chen C Zhang Si-wei SW Wu Zhong-shan ZS Cui Zhi-cheng ZC Melcher Karsten K Zhang Cheng-hai CH Jiang Yi Y Cong Yao Y Xu H Eric HE

Acta pharmacologica Sinica 20150615 8

<h4>Aim</h4>To report our methods for expression and purification of α7 nicotinic acetylcholine receptor (α7-nAChR), a ligand-gated pentameric ion channel and an important drug target.<h4>Methods</h4>α7-nAChRs of 10 different species were cloned into an inducible BacMam vector with an N-terminal tag of a tandem maltose-binding protein (MBP) and a TEV cleavage site. This α7-nAChR fusion receptor was expressed in mammalian HEK293F cells and detected by Western blot. The expression was scaled up to ...[more]

PMID: 26073323

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Project description:PurposeThe α7 nicotinic acetylcholine receptor (nAChR) is widely expressed in the nervous system, including in the inner retinal neurons in all species studied to date. Although reductions in the expression of α7 nAChRs are thought to contribute to the memory and visual deficits reported in Alzheimer's disease (AD) and schizophrenia , the α7 nAChR knockout (KO) mouse is viable and has only slight visual dysfunction. The absence of a major phenotypic abnormality may be attributable to developmental mechanisms that serve to compensate for α7 nAChR loss. We hypothesized that the upregulation of genes encoding other nAChR subunits or muscarinic acetylcholine receptor (mAChR) subtypes during development partially accounts for the absence of major deficiencies in the α7 nAChR KO mouse. The purpose of this study was to determine whether the deletion of the α7 nAChR subunit in a mouse model resulted in changes in the regulation of other cholinergic receptors or other ion channels in an α7 nAChR KO mouse when compared to a wild-type (WT) mouse.MethodsTo examine gene expression changes, we employed a quantitative real-time polymerase chain reaction (qPCR) using whole retina RNA extracts as well as RNA extracted from selected regions of the retina. These extracts were collected using laser capture microdissection (LCM). The presence of acetylcholine receptor (AChR) subunit and subtype proteins was determined via western blotting. To determine any differences in the number and distribution of choline acetyltransferase (ChAT) amacrine cells, we employed wholemount and vertical immunohistochemistry (IHC) and cell counting. Additionally, in both WT and α7 nAChR KO mouse retinas, the distribution of the nAChR subunit and mAChR subtype proteins were determined via IHC for those KO mice that experienced mRNA changes.ResultsIn the whole retina, there was a statistically significant upregulation of α2, α9, α10, β4, nAChR subunit, and m1 and m4 mAChR subtype transcripts in the α7 nAChR KO mice. However, the retinal layers showed complex patterns of transcript expression. In the ganglion cell layer (GCL), m2 and m4 mAChR subtype transcripts were significantly upregulated, while β3 and β4 nAChR subunit transcripts were significantly downregulated. In the inner portion of the inner nuclear layer (iINL), α2, α9, β4, nAChR subunit, and m3 and m4 mAChR subtype transcripts were significantly downregulated. In the outer portion of the inner nuclear layer (oINL), β2, β4, and m4 AChR subunit transcripts were significantly upregulated. Western blot experiments confirmed the protein expression of α3-α5 and α9-containing nAChR subunits and m1-m2 mAChR subtypes in mouse retinas. IHC results supported many of the mRNA changes observed. Finally, this is the first report of α9 and α10 nAChR subunit expressions in the retina of any species.ConclusionsRather than a simple upregulation of a single AChR subunit or subtype, the absence of the α7 nAChR in the KO mice was associated with complex layer-specific changes in the expression of AChR subunits and subtypes.

Project description:Purpose: Eye drop application of PNU-282987, an α7 nAChR agonist, causes regeneration through activation of Müller glia (MG) in the adult rodent retina. The goals of this study are to examine the transcrptional changes through RNA-seq after treatment with PNU-282987 in mammalian MG cell culture that result in dedifferentiated MG that have a retinal progentior cell(RPC)-like fate and to vallidate RNA-seq differentially expressed genes with qRT-PCR and immunocytochemistry. Methods: Retinal pigment epithelium (RPE-J) cells were treated with PNU-282987 for 24 hours and then washed extensivley. These treated RPE cells were co-cultured via a transwell system with MG (rMC-1) cell culture for 0, 12, 24 and 48 hours. RNA was extracted from MG using Zymo Direct-zol RNA miniprep kit and mRNA proflies were generated by GeneWiz with the Illumina NextSeq 550 high-output platform. Results: Using an optimized data analysis workflow, GeneWiz mapped about 30 million sequence reads per sample to the rat genome (Rnor6.0) and identified over 15,000 transcripts. Deseq2 analysis was performed to determine log 2 fold changes in gene expression compared to DMSO control and found 1050 significantly differentially expressed genes and 12 of these were validated with qRT–PCR to compare trends. Notably, the HB-EGF/Ascl1/Lin28 pathway was signficantly upregulated. ICC showed expression of RPC genes that were also seen in the RNA-seq profile. Conclusions: PNU-282987 activation of RPE causes signficant transcript profile changes in MG cell culture that leads to upregulation of the HB-EGF/Ascl1/Lin28 pathway that was seen with RNA-seq profiles and validated with qRT-PCR. Further, these gene expression changes lead to dedifferentation of MG to an RPC-like fate and expression of several retinal RPC markers were found in RNA-seq data set and was validated with immunocytochemistry. Our study is the first to show that activaiton of the α7 nAChR on RPE can lead to MG dedifferentiation and that the HB-EGF/Ascl1/Lin28 pathway is upregulated in these cells.

Project description:ImportanceStudies using human postmortem tissue and imaging with positron emission tomography (PET) support a low hippocampal availability of the α7 nicotinic acetylcholine receptor (α7-nAChR) in psychotic conditions, particularly in schizophrenia or schizoaffective disorder (nonaffective psychosis). If validated further, the finding may have implications for clinical diagnosis and treatment.ObjectiveTo test for lower availability of the α7-nAChR in the hippocampus of individuals with recent-onset psychosis compared with healthy control individuals and its association with lower cognitive performance or higher psychotic symptom burden within recent-onset psychosis.Design, setting, and participantsIn this cross-sectional study, healthy individuals without history of psychosis and patients within 10 years of a first onset of psychotic disorder were recruited from the greater Baltimore, Maryland, and Washington, DC, area. Fluorine 18-labeled ASEM ([18F] ASEM) PET data were acquired from participants enrolled between March 1, 2014, and July 31, 2023, from an academic research institution. Data acquired between March 1, 2014, and January 31, 2018 (n = 26), were published as a pilot study and were combined with new data acquired between January 1, 2019, and July 31, 2023 (n = 33).Main outcome and measuresRegional [18F]ASEM total distribution volume (VT) that measures α7-nAChR availability, global cognition composite score, and total scores on the Scale for the Assessment of Positive Symptoms and the Scale for the Assessment of Negative Symptoms.ResultsA total of 59 participants (30 women [51%]; mean [SD] age, 25.5 [5.2] years), including 35 with recent-onset psychosis and 24 healthy controls, completed the study. In age-adjusted analyses, lower hippocampal [18F]ASEM VT was found in individuals with recent-onset psychosis (mean [SE], 17.87 [0.60]) compared with healthy controls (mean [SE], 19.82 [0.73]) (P = .04). In addition, [18F]ASEM VT was lower in individuals with nonaffective psychosis (mean [SE], 16.30 [0.83]) compared with healthy controls (P = .006) or those with affective psychosis (mean [SE], 19.34 [0.80]) (P = .03). Across recent-onset psychosis and after controlling for age, lower hippocampal [18F]ASEM VT was associated with more positive (r = -0.44; P = .009) but not negative symptoms, and higher hippocampal VT was associated with better global cognition composite score (r = 0.38; P = .03).Conclusions and relevanceIn this cross-sectional study of individuals with recent-onset psychosis compared with healthy controls, a lower hippocampal α7-nAChR availability was found in recent-onset psychosis, and its availability was lower in those with nonaffective vs affective psychosis. Further study of the association between low availability of the α7-nAChR and recent-onset psychosis is warranted toward informing diagnostic or therapeutic strategies related to these findings.

Dataset Information

Crystallization scale purification of ?7 nicotinic acetylcholine receptor from mammalian cells using a BacMam expression system.

Publications

Crystallization scale purification of α7 nicotinic acetylcholine receptor from mammalian cells using a BacMam expression system.

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