Neuronal nicotinic receptor β2 and β4 subunits confer large differences in agonist binding affinity

We used equilibrium binding analysis to characterize the agonist binding properties of six different rat neuronal nicotinic receptor subunit combinations expressed in Xenopus laevis oocytes. The α4β2 receptor bound [³H]cytisine with a K(dapp) of 0.74 ± 0.14 nM. The rank order of K(iapp) values of additional nicotinic ligands, determined in competition assays, was cytisine < nicotine < acetylcholine < carbachol < curare. These pharmacological properties of α4β2 expressed in oocytes are comparable to published values for the high affinity cytisine binding site in rat brain (α4β2), demonstrating that rat neuronal nicotinic receptors expressed in X. laevis oocytes display appropriate pharmacological properties. Use of [³H]epibatidine allowed detailed characterization of multiple neuronal nicotinic receptor subunit combinations. K(dapp) values for [³H]epibatidine binding were 10 pM for α2β2, 87 pM for α2β4, 14 pM for α3β2, 300 pM for α3β4, 30 pM for α4β2, and 85 pM for α4β4. Affinities for six additional agonists (acetylcholine, anabasine, cytisine, 1,1-dimethyl-4- phenylpiperazinium, lobeline, and nicotine) were determined in competition assays. The β2-containing receptors had consistently higher affinities for these agonists than did β4-containing receptors. Particularly striking examples are the affinities displayed by α2β2 and α2β4, which differ in 1,1-dimethyl-4-phenylpiperazinium, nicotine, lobeline, and acetylcholine affinity by 120-, 86-, 85-, and 61-fold, respectively. Although smaller differences in affinity could be ascribed to different α subunits, the major factor in determining agonist affinity was the nature of the β subunit.