| The nicotinic acetylcholine receptors are pentameric ligand-gated ion channels that mediate fast synaptic transmission in the brain and peripheral nervous system. After an introduction (Chapter 1), Chapter 2 describes my development of techniques to combine single-channel and whole-cell recording with nonsense suppression. Having established the feasibility of the combined use of single-channel and whole-cell recording, in Chapter 3 we developed a method to identify the functional interactions of amino acids that are physically far apart in a protein. This is fundamentally a whole-cell recording method to find allosteric interactions in ion channels. The significance of this method is strongly supported by single-channel measurements. Additionally, the relationship between the single-channel gating equilibrium constant, theta, and the whole-cell measurement of EC50 is considered.;In Chapter 4, I describe my progress towards measuring the channel opening rate of the fetal and adult muscle-type nicotinic acetylcholine receptors. Multiple different agonists are used, including acetylcholine, choline, and tetramethylammonium. Single-channel data are reported for the wild-type receptors as well as for receptors with the unnatural amino acid 5-F-Trp (monofluoro-Trp). Data are reported for multiple concentrations for a mutated fetal nAChR, and QuB is used to fit various possible models and estimate theta for this mutant.;A major aim of this dissertation was to use single-molecule studies of ion channels expressing unnatural amino acids to provide even more convincing evidence for cation-pi interactions at the binding sites of ligand-gated ion channels, specifically the neuronal nicotinic acetylcholine receptor. Chapter 5 describes the combined application of single-channel, whole-cell, and unnatural amino acid mutagenesis to the specific question of how two molecules---nicotine and ChantixRTM (varenicline)---bind to the alpha4beta2 brain receptor.;In Chapter 6, I describe single-channel experiments that establish a method for distinguishing between the two known stoichiometries of the wild type alpha4beta2 brain receptor. Specifically, I identify a difference in the rectification properties of the high and low affinity receptors. |
