Pharmacological and immunochemical characterization of α2* nicotinic acetylcholine receptors (nAChRs) in mouse brain

Abstract

Aim: α2 nAChR subunit mRNA expression in mice is most intense in the olfactory bulbs and interpeduncular nucleus. We aimed to investigate the properties of α2* nAChRs in these mouse brain regions. Methods: α2 nAChR subunit-null mutant mice were engineered. Pharmacological and immunoprecipitation studies were used to determine the composition of α2 subunit-containing (α2*) nAChRs in these two regions. Results: [ 125 I]Epibatidine (200 pmol/L) autoradiography and saturation binding demonstrated that α2 deletion reduces nAChR expression in both olfactory bulbs and interpeduncular nucleus (by 4.8±1.7 and 92±26 fmol·mg -1 protein, respectively). Pharmacological characterization using the β2-selective drug A85380 to inhibit [ 125 I]epibatidine binding proved inconclusive, so immunoprecipitation methods were used to further characterize α2* nAChRs. Protocols were established to immunoprecipitate β2 and β4 nAChRs. Immunoprecipitation specificity was ascertained using tissue from β2-and β4-null mutant mice, and efficacy was good (>90% of β2* and >80% of β4* nAChRs were routinely recovered). Conclusion:Immunoprecipitation experiments indicated that interpeduncular nucleus α2* nAChRs predominantly contain β2 subunits, while those in olfactory bulbs contain mainly β4 subunits. In addition, the immunoprecipitation evidence indicated that both nuclei, but especially the interpeduncular nucleus, express nAChR complexes containing both β2 and β4 subunits.

α-Conotoxins Identify the α3β4* Subtype as the Predominant Nicotinic Acetylcholine Receptor Expressed in Human Adrenal Chromaffin Cells

“…Examples of current traces showing the inhibition produced by the two a-Ctxs are shown in Fig. 4, B and C. Most heteromeric human nAChR subtypes, with the exception of the a9a10 subtype, fall into two categories that contain either b2 or b4 subunits, but mixed subtypes that contain both b2 and b4 ligand-binding sites have been shown to be present in several brain regions in rodents (Turner and Kellar, 2005;Azam and McIntosh, 2006;Grady et al, 2009;Whiteaker et al, 2009). Binding of a single a-Ctx molecule to a nAChR subunit interface is sufficient to block the allosteric transitions required for ion channel opening (Talley et al, 2006), and binding to a target site can occur with high affinity without regard to composition of other binding sites in the receptor complex.…”

Skeletal parasympathetic innervation communicates central IL-1 signals regulating bone mass accrual

“…Because the α 2 -and β 2 nAChR subunits can form receptor complexes (28), and because α-subunits are essential components of nAChRs, we analyzed the skeletal phenotype of α 2 nAChR −/− mice (29). These mice show a marked low bone mass ( Fig.…”

“…) and mice deficient of the α 2 nAChR subunit were reported previously (23,29). The latter were kindly provided by Jim Boulter, Department of Psychiatry and Biobehavioral Sciences, University of California, Los Angeles, CA).…”

Differential expression of the beta4 neuronal nicotinic receptor subunit affects tolerance development and nicotinic binding sites following chronic nicotine treatment

“…Deletion of the β4 subunit had little effect on these sites in either superior or inferior colliculus, but reduced them in olfactory bulb, indicating the presence of a relatively complex set of receptors in this region. Such complexity could be expected owing to the diversity of nAChR subunit mRNA expression in olfactory bulb a brain region that includes relatively high levels of mRNA encoding α2, α3, β2 and β4 nAChR subunits (Dineley-Miller and Patrick, 1992; Wada et al, 1989) and the expression of α2*-nAChR in olfactory bulb has been demonstrated (Whiteaker et al, 2009). Characterization of nAChR expression in superior colliculus has revealed the existence of significant receptor populations that include α3 and α6 subunits (McClure-Begley et al, 2014).…”