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The ?3 subunit of the nicotinic acetylcholine receptor: Modulation of gene expression and nicotine consumption.

ABSTRACT: Genetic factors explain approximately half of the variance in smoking behaviors, but the molecular mechanism by which genetic variation influences behavior is poorly understood. SNPs in the putative promoter region of CHRNB3, the gene that encodes the ?3 subunit of the nicotinic acetylcholine receptor (nAChR), have been repeatedly associated with nicotine behaviors. In this work we sought to identify putative function of three SNPs in the promoter region of CHRNB3 on in vitro gene expression. Additionally, we used ?3 null mutant mice as a model of reduced gene expression to assess the effects on nicotine behaviors. The effect of rs13277254, rs6474413, and rs4950 on reporter gene expression was examined using a luciferase reporter assay. A major and minor parent haplotype served as the background on which alleles at the three SNPs were flipped onto different backgrounds (e.g. minor allele on major haplotype background). Constructs were tested in three human cell lines: BE(2)-C, SH-SY5Y and HEK293T. In all cell types the major haplotype led to greater reporter gene expression compared to the minor haplotype, and results indicate that this effect is driven by rs6474413. Moreover, mice lacking the ?3 subunit showed reduced voluntary nicotine consumption compared that of wildtype animals. These data provide evidence that the protective genetic variant at rs6474413 identified in human genetic studies reduces gene expression and that decreased ?3 gene expression in mice reduces nicotine intake. This work contributes to our understanding of the molecular mechanisms that contribute to the human genetic associations of tobacco behaviors.

SUBMITTER: Kamens HM

PROVIDER: S-EPMC4655151 | biostudies-literature | 2015 Dec

REPOSITORIES: biostudies-literature

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The β3 subunit of the nicotinic acetylcholine receptor: Modulation of gene expression and nicotine consumption.

Kamens Helen M HM Miyamoto Jill J Powers Matthew S MS Ro Kasey K Soto Marissa M Cox Ryan R Stitzel Jerry A JA Ehringer Marissa A MA

Neuropharmacology 20150825

Genetic factors explain approximately half of the variance in smoking behaviors, but the molecular mechanism by which genetic variation influences behavior is poorly understood. SNPs in the putative promoter region of CHRNB3, the gene that encodes the β3 subunit of the nicotinic acetylcholine receptor (nAChR), have been repeatedly associated with nicotine behaviors. In this work we sought to identify putative function of three SNPs in the promoter region of CHRNB3 on in vitro gene expression. Ad ...[more]

PMID: 26318101

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Project description:The CHRNA5-CHRNA3-CHRNB4 gene cluster, encoding the α5, α3, and β4 nicotinic acetylcholine receptor (nAChR) subunits, has been linked to nicotine dependence. The habenulo-interpeduncular (Hb-IPN) tract is particularly enriched in α3β4 nAChRs. We recently showed that modulation of these receptors in the medial habenula (MHb) in mice altered nicotine consumption. Given that β4 is rate-limiting for receptor activity and that single nucleotide polymorphisms (SNPs) in CHRNB4 have been linked to altered risk of nicotine dependence in humans, we were interested in determining the contribution of allelic variants of β4 to nicotine receptor activity in the MHb. We screened for missense SNPs that had allele frequencies >0.0005 and introduced the corresponding substitutions in Chrnb4. Fourteen variants were analyzed by co-expression with α3. We found that β4A90I and β4T374I variants, previously shown to associate with reduced risk of smoking, and an additional variant β4D447Y, significantly increased nicotine-evoked current amplitudes, while β4R348C, the mutation most frequently encountered in sporadic amyotrophic lateral sclerosis (sALS), showed reduced nicotine currents. We employed lentiviruses to express β4 or β4 variants in the MHb. Immunoprecipitation studies confirmed that β4 lentiviral-mediated expression leads to specific upregulation of α3β4 but not β2 nAChRs in the Mhb. Mice injected with the β4-containing virus showed pronounced aversion to nicotine as previously observed in transgenic Tabac mice overexpressing Chrnb4 at endogenous sites including the MHb. Habenular expression of the β4 gain-of-function allele T374I also resulted in strong aversion, while transduction with the β4 loss-of function allele R348C failed to induce nicotine aversion. Altogether, these data confirm the critical role of habenular β4 in nicotine consumption, and identify specific SNPs in CHRNB4 that modify nicotine-elicited currents and alter nicotine consumption in mice.

Dataset Information

The ?3 subunit of the nicotinic acetylcholine receptor: Modulation of gene expression and nicotine consumption.

Publications

The β3 subunit of the nicotinic acetylcholine receptor: Modulation of gene expression and nicotine consumption.

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