Agonist Binding Studies at Two Subtypes of the Nicotinic Acetylcholine Receptor Involved in Parkinson’s Disease and Addiction

Citation

Post, Michael Robert (2016) Agonist Binding Studies at Two Subtypes of the Nicotinic Acetylcholine Receptor Involved in Parkinson’s Disease and Addiction. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/Z9GT5K50. https://resolver.caltech.edu/CaltechTHESIS:06072016-160630609

Abstract

Neuronal nicotinic acetylcholine receptors (nAChR) consist of pentameric ligand gated ion channels that typically regulate the release of neurotransmitter. This group of receptors is made of many subunits that combine into pentamers to form different subtypes, with each subtype having a unique pharmacology, function, and localization in the nervous system. The α6β2 subtype is found predominantly in the dopaminergic pathways in the brain, and is therefore a promising target for addiction and Parkinson’s disease. A major goal in treating these disorders is to develop subtype-selective agonists, and advanced knowledge of the binding site which sits at the α6-β2 subunit interface is critical. This thesis dissertation describes high precision, chemical scale structure-function studies designed to probe specific interactions between a variety of agonists and the amino acids which make up the α6β2 binding site.

Before these studies, which utilize nonsense suppression-based non-canonical amino acid mutagenesis, could be conducted, a heterologous expression system for α6β2 had to be developed.

Chapter 2 details four reporter mutations that allow high expression levels of α6β2 in Xenopus oocytes. Further work presented in this chapter characterizes a variety of compounds at this subtype including acetylcholine, the endogenous agonist, nicotine, and TC299423, a promising drug candidate designed to be α6-selective.

Chapters 3 and 4 discuss the structure-function studies used to probe for binding interactions of acetylcholine, nicotine, and TC299423 with the α6-β2 interface. Fluorination series were executed to probe for cation-π interactions with TrpB, TyrA, and TyrC2, all sites of the α6 face. Of the nine possible agonist-side chain interactions, the only functionally important cation-π interaction was found between acetylcholine and TrpB, suggesting the subtype has a unique pharmacology. Studies utilizing α-hydroxy acids were then performed to determine whether these agonists make a functional hydrogen bond between their amine NH and the backbone carbonyl associated with TrpB. Here, nicotine was found to make a strong hydrogen bond, whose energy was quantified via double-mutant cycle analysis, but TC299423 was not.

Chapter 5 further explores TC299423 at the α4β2 subtype. Experiments here showed that TC299423 makes a dual cation-π interaction with both TrpB and TyrC2. Further studies revealed this dual cation-π effect to be true for several secondary amines, and a structure-function study with nornicotine established this as a general feature for secondary amines.

Chapter 6 describes work done to probe for a hydrogen bond between the indole NH of α4 TrpB and a backbone carbonyl associated with L119 on the β2 subunit. This study required development of a new strategy to probe for hydrogen bonds as the amino acid sequence does not allow for α-hydroxy substitution. Instead, a fluorinated side chain strategy was used to inductively attenuate the hydrogen bond accepting ability of the carbonyl, and it proved the α4-β2 interfacial hydrogen prediction false.

Finally two appendices suggest possible avenues to explore with the new α6β2 expression system. Appendix A describes work done to determine whether there is cross-talk between α6β2 and P2X receptors. Appendix B details initial investigations on the effects of ethanol and other alcohols on the function of α6β2.

Thesis Files