Pharmacological properties of alpha4beta2 and alpha3beta4 nicotinic acetylcholine receptors: Ligand binding, activation, desensitization, and interspecies differences.

摘要

Smoking cessation medications using nicotinic ligands, including nicotine, cytisine, and varenicline, have traditionally revolved around selective activation of alpha4beta2 nicotinic acetylcholine receptors (nAChRs). However, nAChR desensitization may be the primary action of nicotine and may be a viable approach for smoking cessation medication. The alpha3beta4 nAChR subtype has been implicated in side effects of nicotinic drugs as well as nicotine addiction. The purpose of the work here is to investigate pharmacological properties of these two nAChR subtypes, with a focus on mechanisms of ligand binding, activation, desensitization, and interspecies differences between human and rat receptors.;The first aim of this dissertation was to dissect the properties of six ligands (nicotine, cytisine, sazetidine-A, varenicline, epibatidine, and 5-I-A85380) at alpha4beta2 nAChRs. We developed a method for determining the dissociation rates of unlabeled ligands. These ligands are more potent at desensitizing than at activating alpha4beta2 nAChRs. A correlation was found between duration of desensitization and binding affinity.;The second aim was to develop methods for using microfluidic laminar stream solution exchange (MLSSE) to study nAChRs expressed in HEK293 cell lines. The variables considered in this technique were drug exposure time and washout time. We demonstrated that MLSSE can be applied to a broad range of nAChRs.;The final aim studied interspecies differences in alpha3beta4 drug affinity. All tested drugs besides acetylcholine have higher potency for human alpha3beta4 nAChRs than rat nAChRs. AT-1001, a new high affinity alpha3beta4 ligand, does not select for desensitization over activation in alpha3beta4 or alpha4beta2 nAChRs. Furthermore, the differences in affinity of AT-1001 between alpha3beta4 and alpha4beta2 nAChRs result in longer desensitization in alpha3beta4 nAChRs compared to alpha4beta2 nAChRs. This is consistent with the affinity of a ligand for a receptor subtype being the major determinant for desensitization length.;The investigations in my dissertation contribute to a better understanding of the pharmacological properties of alpha4beta2 and alpha3beta4 nAChRs. The methods developed will help in future studies. Moreover, the results provide important insight in developing new nicotinic therapeutics based on nAChR desensitization and indicate that investigators should be aware of species differences in studying nicotinic ligands.