Molecular mechanisms regulating neuronal nicotinic acetylcholine receptor function and expression

Neuronal nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels involved in a variety of biological processes; they play a key role in synaptic transmission. Their malfunctioning leads to a variety of diseases, and is the driving force for numerous investigations. This work is focused on the alpha4beta2 nAChR subtype, which plays a crucial role in nicotine addiction. The goal of this thesis dissertation is to understand the molecular mechanisms by which chronic nicotine exposure affects the function and expression of the alpha4beta2 nAChR subtype. The function and expression of alpha4beta2 nAChRs was evaluated using electrophysiological and molecular biology techniques. The first chapter of this dissertation provides a general review of the current knowledge of nicotinic acetylcholine receptors. The second chapter defines a new role for phosphorylation residues on the cytoplasmic loop of the alpha4 nAChR subunit. Eleven putative phosphorylation sites were mutated to alanine and aspartic acid to abolish or mimic phosphorylation, respectively. These mutations were expressed in Xenopus laevis oocytes to assess channel function and expression. The most significant results were the discovery of four mutations with an enhanced sensitivity to nicotine and a nicotine-insensitive mutation. The third chapter examined the effects of a long-term treatment (24hrs) with a recently approved smoking cessation drug, varenilcine, on the alpha4beta2 nAChR subtype. The study revealed two significant results: valerinicline down-regulated alpha4beta2 nAChR expression and down-regulated its functional response. Specifically varenicline abolishes fast agonist activation of alpha4beta2 nAChRs in vitro. Collectively, these findings provide a new perspective for the design of smoking cessation drugs.