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 alpha 4 beta 2 receptor bound [H-3]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 alpha 4 beta 2 expressed in oocytes are comparable to published values for the high affinity cytisine binding site in rat brain (alpha 4 beta 2), demonstrating that rat neuronal nicotinic receptors expressed in X. laevis oocytes display appropriate pharmacological properties. Use of [H-3]epibatidine allowed detailed characterization of multiple neuronal nicotinic receptor subunit combinations. K-dapp values for [H-3]epibatidine binding were 10 pM for alpha 2 beta 2, 87 pM for alpha 2 beta 4, 14 pM for alpha 3 beta 2, 300 pM for alpha 3 beta 4, 30 pM for alpha 4 beta 2, and 85 pM for alpha 4 beta 4. Affinities for six additional agonists (acetylcholine, anabasine, cytisine, 1,1-dimethyl-4-phenylpiperazinium, lobeline, and nicotine) were determined in competition assays. The beta 2-containing receptors had consistently higher affinities for these agonists than did beta 4-containing receptors. Particularly striking examples are the affinities displayed by alpha 2 beta 2 and alpha 2 beta 4, 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 alpha subunits, the major factor in determining agonist affinity was the nature of the beta subunit.