Habenular expression of rare missense variants of the β4 nicotinic receptor subunit alters nicotine consumption

The CHRNA5-CHRNA3-CHRNB4 gene cluster, encoding the alpha 5, alpha 3, and beta 4 nicotinic acetylcholine receptor (nAChR) subunits, has been linked to nicotine dependence. The habenulo-interpeduncular (Hb-IPN) tract is particularly enriched in alpha 3 beta 4 nAChRs. We recently showed that modulation of these receptors in the medial habenula (MHb) in mice altered nicotine consumption. Given that beta 4 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 beta 4 to nicotine receptor activity in the MHb. We screened for missense SNPs that had allele frequencies >0.0005 and introduced the corresponding substitutions in Chmb4. Fourteen variants were analyzed by co-expression with alpha 3. We found that beta 4A901 and 134T3741 variants, previously shown to associate with reduced risk of smoking, and an additional variant beta 4D447Y, significantly increased nicotine-evoked current amplitudes, while beta 4R348C, the mutation most frequently encountered in sporadic amyotrophic lateral sclerosis (sALS), showed reduced nicotine currents. We employed lentiviruses to express beta 4 or beta 4 variants in the MHb. lmmunoprecipitation studies confirmed that 134 lentiviral-mediated expression leads to specific upregulation of alpha 3 beta 4 but not beta 2 nAChRs in the Mhb. Mice injected with the beta 4-containing virus showed pronounced aversion to nicotine as previously observed in transgenic Tabac mice overexpressing Chmb4 at endogenous sites including the MHb. Habenular expression of the beta 4 gain-of-function allele T374I also resulted in strong aversion, while transduction with the beta 4 loss-of function allele R348C failed to induce nicotine aversion. Altogether, these data confirm the critical role of habenular beta 4 in nicotine consumption, and identify specific SNPs in CHRNB4 that modify nicotine-elicited currents and alter nicotine consumption in mice.