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Nicotinic acetylcholine receptor alpha7 subunits with a C2 cytoplasmic loop yellow fluorescent protein insertion form functional receptors.

ABSTRACT: Several nicotinic acetylcholine receptor (nAChR) subunits have been engineered as fluorescent protein (FP) fusions and exploited to illuminate features of nAChRs. The aim of this work was to create a FP fusion in the nAChR alpha7 subunit without compromising formation of functional receptors.A gene construct was generated to introduce yellow fluorescent protein (YFP), in frame, into the otherwise unaltered, large, second cytoplasmic loop between the third and fourth transmembrane domains of the mouse nAChR alpha7 subunit (alpha7Y). SH-EP1 cells were transfected with mouse nAChR wild type alpha7 subunits (alpha7) or with alpha7Y subunits, alone or with the chaperone protein, hRIC-3. Receptor function was assessed using whole-cell current recording. Receptor expression was measured with (125)I-labeled alpha-bungarotoxin (I-Bgt) binding, laser scanning confocal microscopy, and total internal reflectance fluorescence (TIRF) microscopy.Whole-cell currents revealed that alpha7Y nAChRs and alpha7 nAChRs were functional with comparable EC(50) values for the alpha7 nAChR-selective agonist, choline, and IC(50) values for the alpha7 nAChR-selective antagonist, methyllycaconitine. I-Bgt binding was detected only after co-expression with hRIC-3. Confocal microscopy revealed that alpha7Y had primarily intracellular rather than surface expression. TIRF microscopy confirmed that little alpha7Y localized to the plasma membrane, typical of alpha7 nAChRs.nAChRs composed as homooligomers of alpha7Y subunits containing cytoplasmic loop YFP have functional, ligand binding, and trafficking characteristics similar to those of alpha7 nAChRs. alpha7Y nAChRs may be used to elucidate properties of alpha7 nAChRs and to identify and develop novel probes for these receptors, perhaps in high-throughput fashion.

SUBMITTER: Murray TA

PROVIDER: S-EPMC4002364 | biostudies-other | 2009 Jun

REPOSITORIES: biostudies-other

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Project description:We and others have shown that one of the mechanisms of growth regulation of small cell lung cancer cell lines and cultured pulmonary neuroendocrine cells is by the binding of agonists to the alpha7 neuronal nicotinic acetylcholine receptor. In addition, we have shown that the nicotine-derived carcinogenic nitrosamine, 4(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), is a high affinity agonist for the alpha7 nicotinic acetylcholine receptor. In the present study, our goal was to determine the extent of alpha7 mRNA and protein expression in the human lung.Experiments were done using reverse transcription polymerase chain reaction (RT-PCR), a nuclease protection assay and western blotting using membrane proteins.We detected mRNA for the neuronal nicotinic acetylcholine receptor alpha7 receptor in seven small cell lung cancer (SCLC) cell lines, in two pulmonary adenocarcinoma cell lines, in cultured normal human small airway epithelial cells (SAEC), one carcinoid cell line, three squamous cell lines and tissue samples from nine patients with various types of lung cancer. A nuclease protection assay showed prominent levels of alpha7 in the NCI-H82 SCLC cell line while alpha7 was not detected in SAEC, suggesting that alpha7 mRNA levels may be higher in SCLC compared to normal cells. Using a specific antibody to the alpha7 nicotinic receptor, protein expression of alpha7 was determined. All SCLC cell lines except NCI-H187 expressed protein for the alpha7 receptor. In the non-SCLC cells and normal cells that express the alpha7 nAChR mRNA, only in SAEC, A549 and NCI-H226 was expression of the alpha7 nicotinic receptor protein shown. When NCI-H69 SCLC cell line was exposed to 100 pm NNK, protein expression of the alpha7 receptor was increased at 60 and 150 min.Expression of mRNA for the neuronal nicotinic acetylcholine receptor alpha7 seems to be ubiquitously expressed in all human lung cancer cell lines tested (except for NCI-H441) as well as normal lung cells. The alpha7 nicotinic receptor protein is expressed in fewer cell lines, and the tobacco carcinogen NNK increases alpha7 nicotinic receptor protein levels.

Project description:ObjectiveThe central nervous system can regulate peripheral inflammation, but the efferent neuronal routes and the mediators remain poorly defined. One candidate is the cholinergic pathway, which releases acetylcholine (ACh). This neurotransmitter can bind to the alpha7 cholinergic receptor (alpha7R) expressed by nonneuronal cells and reduce inflammation. To test this possibility, we evaluated the expression of alpha7R and its potential role as a target in rheumatoid arthritis (RA).MethodsThe expression of alpha7R in human synovium and fibroblast-like synoviocytes (FLS) was determined using immunohistochemical, Western blot, and quantitative polymerase chain reaction (PCR) analyses. The effects of ACh in vitro were determined in interleukin-1 (IL-1)-stimulated FLS using immunoassays for protein, quantitative PCR for messenger RNA (mRNA), luciferase reporter constructs for IL-6 and NF-kappaB promoter activity, and electrophoretic mobility shift assays. Expression of alpha7R was knocked down with small interfering RNA (siRNA) or was inhibited with the selective alpha7R antagonist methyllycaconitine (MLA).ResultsProtein and mRNA for alpha7R were demonstrated in RA and osteoarthritis synovium and cultured synoviocytes. Expression in synovium was mainly in the intimal lining. ACh significantly reduced the production of IL-6, CXCL8, CCL2, CCL3, CCL5, and granulocyte colony-stimulating factor by IL-1-stimulated FLS. This effect was blocked by the alpha7R antagonist MLA or by using alpha7R siRNA to knock down receptor expression. The selective alpha7R agonist PNU-282,987 decreased the production of IL-6 by IL-1-stimulated FLS. ACh did not reduce IL-6 transcription, but it decreased IL-6 mRNA half-life and reduced IL-6 mRNA steady-state levels.ConclusionThe alpha7 receptor is expressed in the synovium and by synoviocytes. Receptor ligation inhibits cytokine expression in FLS through a posttranscriptional mechanism. Therefore, alpha7R is a potential therapeutic target for inflammatory diseases.

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Nicotinic acetylcholine receptor alpha7 subunits with a C2 cytoplasmic loop yellow fluorescent protein insertion form functional receptors.

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