Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
RIC-3 expression and splicing regulate nAChR functional expression.
Ben-David Y
,
Mizrachi T
,
Kagan S
,
Krisher T
,
Cohen E
,
Brenner T
,
Treinin M
.
???displayArticle.abstract???
BACKGROUND: The nicotinic acetylcholine receptors form a large and diverse family of acetylcholine gated ion channels having diverse roles in the central nervous system. Maturation of nicotinic acetylcholine receptors is a complex and inefficient process requiring assistance from multiple cellular factors including RIC-3, a functionally conserved endoplasmic reticulum-resident protein and nicotinic acetylcholine receptor-specific chaperone. In mammals and in Drosophila melanogaster RIC-3 is alternatively spliced to produce multiple isoforms.
RESULTS: We used electrophysiological analysis in Xenopus laevis oocytes, in situ hybridization, and quantitative real-time polymerase chain reaction assays to investigate regulation of RIC-3's expression and splicing and its effects on the expression of three major neuronal nicotinic acetylcholine receptors. We found that RIC-3 expression level and splicing affect nicotinic acetylcholine receptor functional expression and that two conserved RIC-3 isoforms express in the brain differentially. Moreover, in immune cells RIC-3 expression and splicing are regulated by inflammatory signals.
CONCLUSIONS: Regulation of expression level and splicing of RIC-3 in brain and in immune cells following inflammation enables regulation of nicotinic acetylcholine receptor functional expression. Specifically, in immune cells such regulation via effects on α7 nicotinic acetylcholine receptor, known to function in the cholinergic anti-inflammatory pathway, may have a role in neuroinflammatory diseases.
Fig. 1. Effects of different amounts of FL on ACh-stimulated currents through each of three nAChRs: a α4β2; b α3β4; and c α7. Results were normalized to currents recorded in oocytes expressing the respective receptors in the absence of RIC-3 in the same experiment. Each bar represents 10–20 oocytes from 2 to 3 independent X. laevis. The y-axis ordinates are on a log scale. * indicates a p value of less than 0.05; ** indicates a p value of less than 0.01
Fig. 2. Effects of different amounts of TM on ACh-stimulated currents through each of three nAChRs: a α4β2; b α3β4; and c α7. Results were normalized to currents recorded in oocytes expressing the respective receptors in the absence of RIC-3 in the same experiment. Each bar represents 10–20 oocytes from 2 to 3 independent X. laevis. The y-axis ordinates are on a log scale. * indicates a p value of less than 0.05; ** indicates a p value of less than 0.01
Fig. 3. Effects of TM and FL cannot be attributed to expression level. a Representative Western Blot of the myc-tagged transcript-expressing oocytes showing similar expression of FL and TM. A single oocyte’s worth of homogenate prepared from an average of about 5 oocytes, was run in each lane; the Western was repeated twice with nearly identical results; b Electrophysiological recordings of ACh-stimulated currents through α7, co-expressed with different amounts of myc-tagged RIC-3 transcripts. Each bar represents 7–11 oocytes from 2 independent X. laevis. The y-axis ordinates are on a log scale. * indicates a p value of less than 0.05
Fig. 4. RIC-3 expression in mouse brain by in situ hybridization. a Schematic representation of the transcripts encoding the FL and TM isoforms and loci of the isoform-specific probes. Lines, introns, black filled bars coding sequences, white filled bars non-coding (UTRs). Scale bar, 100 bp, size of non-coding sequences is not to scale. b representative image of FL expression in hippocampus. c Representative image of TM expression in hippocampus. d Representative image of FL expression in cerebellum. e Representative image of TM expression in cerebellum
Fig. 5. RIC-3 expression in mouse brain by qRT-PCR. a FL and b TM expression in CNS regions of naïve mice. ** indicates a p value of less than 0.01; *** indicates a p value of less than 0.001 according to Students t-test comparing expression in each tissue to expression in the cerebellum. Expression was tested using qRT-PCR for each isoform in each tissue of 10–12 mice. Expression values are normalized to GAPDH expression and multiplied by a factor of 1,000 for convenience. Cerb = cerebellum; hip = hippocampus; pfc = prefrontal cortex; sc = spinal cord
Fig. 6. RIC-3 expression in a mouse spleen cells, and b RAW264.7 cells at 0, 24 and 48 h following exposure to LPS. * indicates a p value of less than 0.05; ** indicates a p value of less than 0.01 according to two-way ANOVA with Bonferroni correction. Expression was tested using qRT-PCR for each isoform in each of 6 spleens, or from each of three independent RAW264.7 experiments. Expression values are normalized to GAPDH expression and multiplied by a factor of 10,000 for convenience
Alexander,
Ric-3 promotes alpha7 nicotinic receptor assembly and trafficking through the ER subcompartment of dendrites.
2010, Pubmed
Alexander,
Ric-3 promotes alpha7 nicotinic receptor assembly and trafficking through the ER subcompartment of dendrites.
2010,
Pubmed
Ben-Ami,
RIC-3 affects properties and quantity of nicotinic acetylcholine receptors via a mechanism that does not require the coiled-coil domains.
2005,
Pubmed
,
Xenbase
Biala,
The conserved RIC-3 coiled-coil domain mediates receptor-specific interactions with nicotinic acetylcholine receptors.
2009,
Pubmed
Borovikova,
Vagus nerve stimulation attenuates the systemic inflammatory response to endotoxin.
2000,
Pubmed
Boulter,
Alpha 3, alpha 5, and beta 4: three members of the rat neuronal nicotinic acetylcholine receptor-related gene family form a gene cluster.
1990,
Pubmed
Boulter,
Isolation of a cDNA clone coding for a possible neural nicotinic acetylcholine receptor alpha-subunit.
,
Pubmed
Castillo,
Role of the RIC-3 protein in trafficking of serotonin and nicotinic acetylcholine receptors.
2006,
Pubmed
Cohen Ben-Ami,
Receptor and subunit specific interactions of RIC-3 with nicotinic acetylcholine receptors.
2009,
Pubmed
,
Xenbase
Cohn,
Lipopolysaccharide-induced inflammation attenuates taste progenitor cell proliferation and shortens the life span of taste bud cells.
2010,
Pubmed
Cordero-Erausquin,
Nicotinic receptor function: new perspectives from knockout mice.
2000,
Pubmed
Dani,
Nicotinic acetylcholine receptors and nicotinic cholinergic mechanisms of the central nervous system.
2007,
Pubmed
Deneris,
Primary structure and expression of beta 2: a novel subunit of neuronal nicotinic acetylcholine receptors.
1988,
Pubmed
,
Xenbase
Du,
[Regulation of lipopolysaccharide-induced inducible nitric oxide synthase gene expression by protein kinase C].
2002,
Pubmed
Flores,
A subtype of nicotinic cholinergic receptor in rat brain is composed of alpha 4 and beta 2 subunits and is up-regulated by chronic nicotine treatment.
1992,
Pubmed
Goldman,
Members of a nicotinic acetylcholine receptor gene family are expressed in different regions of the mammalian central nervous system.
1987,
Pubmed
Gotti,
Brain nicotinic acetylcholine receptors: native subtypes and their relevance.
2006,
Pubmed
Gu,
Brain α7 Nicotinic Acetylcholine Receptor Assembly Requires NACHO.
2016,
Pubmed
Halevi,
Conservation within the RIC-3 gene family. Effectors of mammalian nicotinic acetylcholine receptor expression.
2003,
Pubmed
,
Xenbase
Halevi,
The C. elegans ric-3 gene is required for maturation of nicotinic acetylcholine receptors.
2002,
Pubmed
,
Xenbase
Kalkman,
Modulatory effects of α7 nAChRs on the immune system and its relevance for CNS disorders.
2016,
Pubmed
Kurzen,
The non-neuronal cholinergic system of human skin.
2007,
Pubmed
Lansdell,
Host-cell specific effects of the nicotinic acetylcholine receptor chaperone RIC-3 revealed by a comparison of human and Drosophila RIC-3 homologues.
2008,
Pubmed
Lv,
Upregulating nonneuronal cholinergic activity decreases TNF release from lipopolysaccharide-stimulated RAW264.7 cells.
2014,
Pubmed
Millar,
Assembly and subunit diversity of nicotinic acetylcholine receptors.
2003,
Pubmed
Nelson,
Alternate stoichiometries of alpha4beta2 nicotinic acetylcholine receptors.
2003,
Pubmed
,
Xenbase
Shteingauz,
The BTB-MATH protein BATH-42 interacts with RIC-3 to regulate maturation of nicotinic acetylcholine receptors.
2009,
Pubmed
Séguéla,
Molecular cloning, functional properties, and distribution of rat brain alpha 7: a nicotinic cation channel highly permeable to calcium.
1993,
Pubmed
,
Xenbase
Tompa,
Structural disorder throws new light on moonlighting.
2005,
Pubmed
Treinin,
RIC-3 and nicotinic acetylcholine receptors: biogenesis, properties, and diversity.
2008,
Pubmed
Vallés,
Chaperoning α7 neuronal nicotinic acetylcholine receptors.
2012,
Pubmed
Wang,
Mouse RIC-3, an endoplasmic reticulum chaperone, promotes assembly of the alpha7 acetylcholine receptor through a cytoplasmic coiled-coil domain.
2009,
Pubmed
Wang,
Nicotinic acetylcholine receptor alpha7 subunit is an essential regulator of inflammation.
2003,
Pubmed
Williams,
Ric-3 promotes functional expression of the nicotinic acetylcholine receptor alpha7 subunit in mammalian cells.
2005,
Pubmed
,
Xenbase
Xu,
Megacystis, mydriasis, and ion channel defect in mice lacking the alpha3 neuronal nicotinic acetylcholine receptor.
1999,
Pubmed
Zheng,
Bergapten prevents lipopolysaccharide mediated osteoclast formation, bone resorption and osteoclast survival.
2014,
Pubmed