Mechanistic Investigations of Receptor Signaling via Canonical and Non-Canonical Amino Acid Mutagenesis

This dissertation primarily describes investigations of the mechanisms by which pentameric ligand-gated ion channels (pLGICs) activate ("gating") using canonical and non-canonical amino acid mutagenesis. Chapter 1 provides an introduction to the systems being studied, their physiological roles, and the techniques that we have used to study them. Chapter 2 describes a series of experiments comparing the roles of amino acid residues proximal to the neurotransmitter binding site in the type 3 serotonin receptor (5-HT3R) to the aligning residues of the muscle-type nicotinic acetylcholine receptor (nAChR). The findings presented in Chapter 3 assess the functional roles of proline residues in the prokaryotic pLGIC, Erwinia ligand-gated ion channel (ELIC). Chapter 4 describes an extensive investigation of salient details of 5-HT3R gating using canonical and non-canonical amino acid mutagenesis of amino acid residues at the interface of the extracellular domain and transmembrane domain of this receptor. Chapter 5 applies a photocrosslinking strategy employing the non-canonical amino acid p-azidophenylalanine to study dimerization and cofactor interactions of the estrogen receptor α.</p