Studies of the Serotonin Type 3A Receptor and the Chemical Preparation of tRNA.

摘要

This thesis describes studies surrounding a ligand-gated ion channel (LGIC): the serotonin type 3A receptor (5-HT3ARR). Structure-function experiments using unnatural amino acid mutagenesis are described, as well as experiments on the methodology of unnatural amino acid mutagenesis. Chapter 1 introduces LGICs, experimental methods, and an overview of the unnatural amino acid mutagenesis.;In Chapter 2, the binding orientation of the clinically available drugs ondansetron and granisetron within 5 HT3AR is determined through a combination of unnatural amino acid mutagenesis and an inhibition based assay. A cation-pi interaction is found for both ondansetron and granisetron with a specific tryptophan residue (Trp183, TrpB) of the mouse 5 HT3 AR, which establishes a binding orientation for these drugs.;In Chapter 3, further studies were performed with ondansetron and granisetron with 5 HT3AR. The primary determinant of binding for these drugs was determined to not include interactions with a specific tyrosine residue (Tyr234, TyrC2). In completing these studies, evidence supporting a cation-pi interaction of a synthetic agonist, meta-chlorophenylbiguanide, was found with TyrC2.;In Chapter 4, a direct chemical acylation strategy was implemented to prepare full-length suppressor tRNA mediated by lanthanum(III) and amino acid phosphate esters. The derived aminoacyl-tRNA is shown to be translationally competent in Xenopus oocytes. Appendix A.1 gives details of a pharmacological method for determining the equilibrium dissociation constant, KB, of a competitive antagonist with a receptor, known as Schild analysis.;Appendix A.2 describes an examination of the inhibitory activity of new chemical analogs of the 5 HT3AR antagonist ondansetron. Appendix A.3 reports an organic synthesis of an intermediate for a new unnatural amino acid. Appendix A.4 covers an additional methodological examination for the preparation of amino-acyl tRNA.