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 ( K D ≈ 0.02 nM) and lower affinity ( K D ≈ 7 nM), which can be further subdivided through inhibition by selected agonists and antagonists. These subsets are differentially affected by targeted deletion of α7, β2 or β4 subunits. Most, but not all, higher and lower affinity binding sites require β2 (Marks, M.J., Whiteaker, P., Collins, A.C., 2006. Deletion of the α7, β2 or β4 nicotinic receptor subunit genes identifies highly expressed subtypes with relatively low affinity for [ 3H]epibatidine. Mol. Pharmacol. 70, 947–959). Effects of functional α4 gene deletion are reported here. Deletion of α4 virtually eliminated cytisine-sensitive, higher-affinity [ 3H]epibatidine binding as did β2 deletion, confirming that these sites are α4β2*-nAChR. Cytisine-resistant, higher-affinity [ 3H]epibatidine binding sites are diverse and some of these sites require α4 expression. Lower affinity [ 3H]epibatidine binding sites are also heterogeneous and can be subdivided into α-bungarotoxin-sensitive and -resistant components. Deleting α4 did not affect the α-bungarotoxin-sensitive component, but markedly reduced the α-bungarotoxin-resistant component. This effect was similar, but not quite identical, to the effect of β2 deletion. This provides the first evidence that lower-affinity epibatidine binding sites in the brain require expression of α4 subunits. The effects of α4 gene targeting on receptor function were measured using a 86Rb + efflux assay. Concentration–effect curves for ACh-stimulated 86Rb + efflux are biphasic (EC 50 values = 3.3 μM and 300 μM). Targeting α4 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 β2. Thus, α4β2*-nAChRs mediate a significant fraction of both phases of ACh stimulated 86Rb + efflux.