Structure-function studies of nicotinic acetylcholine receptors using unnatural amino acids

Abstract

This dissertation primarily describes structure-function studies of the nicotinic acetylcholine receptors (nAChRs). These studies use a combination of unnatural amino acid mutagenesis and electrophysiology to determine the specific molecular interactions required for neurotransmitter binding to nAChRs. Chapter 2 examines the mode of agonist activation for the α4β2 nAChR, the receptor responsible for nicotine addiction. This study investigates the molecular interactions that differentiate the α4β2 receptor from other receptor subtypes and endow it with the ability to mediate nicotine addiction. We report that the high affinity for nicotine at the α4β2 receptor is a result of a strong cation-π interaction and a strengthened backbone hydrogen bond to a conserved tryptophan (TrpB) of this receptor. We also establish that a point mutation just four residues away from TrpB appears to influence the shape of the agonist binding site, such that it can differentiate the agonist binding mode of the α4β2 and muscle-type receptors. Chapter 3 extends studies of the point mutation near TrpB, termed the “loop B glycine.” We examine the muscle-type, α4β2, and α7 subtypes and show that the identity of this residue strongly correlates with agonist potency. Low-potency receptor subtypes have a glycine at the loop B site, while high-potency receptors have a lysine at this site. We establish that mutation of this residue can to convert a low-potency receptor to a high-potency receptor and vice versa. Chapter 4 investigates the agonist binding mechanism of the α4β4 receptor. We show both ACh and nicotine make a strong cation-π interaction to TrpB, and nicotine makes a strong hydrogen bond to the backbone carbonyl of TrpB. Additionally, chimeric β subunits are used to examine the influence of the complementary binding component on receptor pharmacology for the α4β2 and α4β4 receptors. Last, chapter 5 is a methodology-based project focused on optimizing the incorporation of unnatural amino acids into mammalian cells. Using HEK293T cells, we successfully suppressed an amber stop codon using HSAS, an in vivo aminoacylated tRNA. Additional studies will pursue the viability of in vitro aminoacylated tRNAs for nonsense suppression in mammalian cells.