Gene targeting demonstrates that α4 nicotinic acetylcholine receptor subunits contribute to expression of diverse [3H]epibatidine binding sites and components of biphasic 86Rb+ efflux with high and low sensitivity to stimulation by acetylcholine

Abstract

[3H]Epibatidine binds to nAChR subtypes in mouse brain with higher (KD approximately 0.02 nM) and lower affinity (KD approximately 7 nM), which can be further subdivided through inhibition by selected agonists and antagonists. These subsets are differentially affected by targeted deletion of alpha7, beta2 or beta4 subunits. Most, but not all, higher and lower affinity binding sites require beta2 (Marks, M.J., Whiteaker, P., Collins, A.C., 2006. Deletion of the alpha7, beta2 or beta4 nicotinic receptor subunit genes identifies highly expressed subtypes with relatively low affinity for [3H]epibatidine. Mol. Pharmacol. 70, 947-959). Effects of functional alpha4 gene deletion are reported here. Deletion of alpha4 virtually eliminated cytisine-sensitive, higher-affinity [3H]epibatidine binding as did beta2 deletion, confirming that these sites are alpha4beta2*-nAChR. Cytisine-resistant, higher-affinity [3H]epibatidine binding sites are diverse and some of these sites require alpha4 expression. Lower affinity [3H]epibatidine binding sites are also heterogeneous and can be subdivided into alpha-bungarotoxin-sensitive and -resistant components. Deleting alpha4 did not affect the alpha-bungarotoxin-sensitive component, but markedly reduced the alpha-bungarotoxin-resistant component. This effect was similar, but not quite identical, to the effect of beta2 deletion. This provides the first evidence that lower-affinity epibatidine binding sites in the brain require expression of alpha4 subunits. The effects of alpha4 gene targeting on receptor function were measured using a 86Rb+ efflux assay. Concentration-effect curves for ACh-stimulated 86Rb+ efflux are biphasic (EC50 values=3.3 microM and 300 microM). Targeting alpha4 produced substantial gene-dose dependent reductions in both phases in whole brain and in most of the 14 brain regions assayed. These effects are very similar to those following deletion of beta2. Thus, alpha4beta2*-nAChRs mediate a significant fraction of both phases of ACh stimulated 86Rb+ efflux.

Engineered α4β2 nicotinic acetylcholine receptors as models for measuring agonist binding and effect at the orthosteric low-affinity α4–α4 interface

“…In a study by Marks et al (1998), [ 3 H]epibatidine showed monophasic binding to 12 different mouse brain regions, however, displacement with reference compounds including ACh, nicotine and cytisine revealed biphasic displacement properties, suggesting different receptor populations. Similar expression patterns have been observed in rat, human (Houghtling et al, 1995;Davila-Garcia et al, 1997) and mouse brain tissue (Marks et al, 1998) and much effort has been invested in determining which nAChR subpopulations constitute the collective binding using pharmacological methods and gene knockout techniques (Marks et al, 2010(Marks et al, , 2006(Marks et al, , 2007. Approximately 50% of total brain binding consists of high affinity sites (K d~2 0 pM) whereas the other 50% are sites of significantly lower affinity (K d~5 nM).…”

“…Peculiarly, these populations are dependent on expression of b2, a4 or b4 subunits and their expression pattern resembles that of a4b2 receptors (Marks et al, 2010). Several speculations have been put forward to explain the existence of these low affinity a4 and b2 dependent sites including stoichiometry dependency (2a:3b vs. 3a:2b), state dependency (resting vs. desensitized), improperly assembled receptors or complex mixtures of receptors incorporating more than two different subunits (Person and Wells, 2011;Marks et al, 2007). The recent knowledge that 3a:2b receptors harbor an orthosteric aea interface suggests an alternative explanation.…”

Nicotine-Induced Dystonic Arousal Complex in a Mouse Line Harboring a Human Autosomal-Dominant Nocturnal Frontal Lobe Epilepsy Mutation

“…The results show that dopamine release mediated by the ␣CtxMII-resistant subtypes is affected by the S248F mutation, whereas release mediated by the ␣CtxMII-sensitive subtypes does not differ with genotype. 86 Rb ϩ efflux is mediated by two populations of nAChRs, a low-and a highaffinity form, both thought to be ␣4␤2* subtypes (Marks et al, 2007). In addition, […”

Mouse Striatal Dopamine Nerve Terminals Express α4α5β2 and Two Stoichiometric Forms of α4β2*-Nicotinic Acetylcholine Receptors

“…We have developed and characterized an assay that measures a biphasic agonist-stimulated 86 Rb + efflux from mouse brain synaptosomes (Marks et al 1999). Both HS and LS responses are clearly mediated by α4β2* nAChRs, given that all response is absent from synaptosomes obtained from α4 (Marks et al 2007) and β2 (Marks et al 2000) null mutant mice. Some of the HS response is also mediated by the α4α5β2-nAChR, given that α5 null mutation results in a decrease in agonist efficacy for the higher sensitivity efflux (Brown et al 2007).…”