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RIC-3 expression and splicing regulate nAChR functional expression.
Ben-David Y
,
Mizrachi T
,
Kagan S
,
Krisher T
,
Cohen E
,
Brenner T
,
Treinin M
.
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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
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