Expression, pharmacological and physiological properties of nicotinic acetylcholine receptors

Nicotinic acetylcholine receptors are ligand-gated ion channels. These receptors play important roles in physiological as well as pathophysiological processes. The present work was aimed at studying three questions that were centered on pharmacology, expression and physiology of nicotinic receptors. The first chapter describes the results of work that was aimed at understanding the molecular determinants of interaction between α-conotoxin BuIA and complementary subunits of nicotinic acetylcholine receptors. Proline 6 of BuIA was found to be a major determinant of binding to nAChR β2 subunit. Coupling between proline 6 and the residue at 59th position of the β subunit was found to be equal to 2.4 kcal/mol. This work paves the ground for creating selective ligands that discriminate between α3β2 and α3β4 receptors. The second chapter describes the dependence of expression of human α9-containing nicotinic acetylcholine receptors in the Xenopus laevis oocyte expression system on the 5'leader sequence of the α9 subunit. The human α9 subunit was determined to be the limiting factor in the functional expression of α9-containing receptors. The inclusion of the 5'leader from alfalfa mosaic virus before the α9 coding sequence facilitated the expression of human α9 homomeric receptors by ~70 fold and human α9α10 receptors by ~80 fold. As a result, a vector was created that allowed high iv expression levels of α9-containing nAChRs; this advance allows reliable testing of new compounds that target human α9-containing receptors. The third chapter describes results of work aimed at understanding the interaction between rat nicotinic α9α10 and purinergic receptors. Comparison of currents from coactivation of receptors to the predicted currents gave inconclusive results. Comparison of agonist sensitivities for purinergic receptors when receptors are expressed alone and when they are coinjected revealed ~1.6-fold difference in sensitivity, with P2X4 receptors less sensitive to ATP when α9α10 receptors are coexpressed. Interactions between rat α9α10 nicotinic receptor and purinergic P2X7 receptors were also examined and yielded negative results.