Biophysical Studies of Ligand-gated Ion Channels

Author: Wong, Betty Ko

Year: 2017

Degree: Dissertation (Ph.D.)

Advisors: Dougherty, Dennis A.; Lester, Henry A.

Committee Members: Gradinaru, Viviana; Rees, Douglas C.; Dougherty, Dennis A.; Lester, Henry A.

Option: Biochemistry and Molecular Biophysics

DOI: 10.7907/Z9TT4P0Q

Abstract

This dissertation describes building a methodology for and the biophysical studies of ligand-gated ion channels (LGICs).

The primary focus of the first half of this dissertation is on developing a fluorescence-based assay to broadly study LGICs. Chapter 2 describes the site-selective incorporation of a turn-on fluorophore via unnatural amino acid mutagenesis on the mouse muscle-type nicotinic acetylcholine receptor (nAChR) in Xenopus oocytes as a proof-of-principle study. This method has proven to yield very low levels of undesired fluorescent background, which was a problem for previous incorporation techniques. Chapter 3 describes efforts towards imaging this in vivo system using lifetime imaging with efforts hampered by the inability to detect a clear signal. Chapter 4 describes efforts to apply the lifetime imaging approach towards a different system involving 5-HT3 proteins fused to fluorescent proteins in COS-7 cells.

The second half of this dissertation focuses on studies of menthol, a flavorant added to cigarettes that contributes to smoking addiction, as a negative allosteric modulator of the αβ42 nAChR. Chapter 5 reveals the stereochemical effects, or rather lack of, of menthol on the two stoichiometries of the αβ42 receptor. Chapter 6 seeks to identify the residue interactions with menthol of the αβ42 receptor using a combination of computational and experimental studies.

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