Nicotinic acetylcholine receptors (nAChRs), the ligand gated ion channels at which nicotine acts, play an important role in drug abuse and may be central to developing rational approaches for substance abuse treatments. The in vitro characterization of the receptor-ligand interaction is an important step in understanding the action of a ligand in vivo (i.e., for interpretation of the results from the functional studies of receptors). To analyze the ligand-receptor interaction at physiological conditions, a competition receptor assay at 37 degrees C was developed. This assay used [I-125]-5-iodo-A-85380 (5IA), which was developed and characterized by staff of the Neuroimaging Research Branch as a high affinity, selective radioligand for alpha4 beta2 nAChRs, to determine the affinities of 28 nicotinic agonists and antagonists (9 established, 8 previously and 11 recently developed in our chemistry lab) at 23 degrees C and 37 degrees C. The Kd values for these ligands ranged from 0.01 to 25,000 nM. Increasing incubation temperature from 23 to 37 degrees C resulted in variable increases in the observed Kd values (from 1.0 to 3.3-fold) and variable effects on the kinetics of receptor-ligand interaction. For example, the association and dissociation of the receptor-ligand complex for 5IA were approximately 10 times faster at 37 degrees C than that at 23 degrees C, but the rate of [H-3]-cytisine binding kinetics increased only 3.5 fold. This variability in effects of temperature on receptor binding properties suggests that characterization of ligand-receptor interaction at physiological temperature may better predict the in vivo properties of the ligands and may be important in the interpretation of results from the functional studies of alpha4 beta2 nAChRs. Previous studies from Abbott Laboratories, where Me-p-PVC (5-(2-(4-pyridinyl)vinyl)-6-chloro-3-(1-methyl-2-(S)-pyrrolidinylmethoxy)pyridine) was developed, demonstrated that this compound had high affinity for alpha4beta2 nicotinic acetylcholine receptors (nAChRs) and attenuated nicotine-induced efflux of 86Rb from IMR-32 cells, contained alpha3beta4 nAChRs. In vitro binding studies with Me-p-PVC showed that its affinity for alpha4beta2 nAChRs exceeded that for the alpha3beta4 receptor subtype by a factor of 3,000 (Kd ca. 0.025 and 90 nM, respectively).
