Structure-Function Studies of Nicotinic Acetylcholine Receptors Using Unnatural Amino Acids

Citation

Xiu, Xinan Joanne (2008) Structure-Function Studies of Nicotinic Acetylcholine Receptors Using Unnatural Amino Acids. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/HWGS-7Z13. https://resolver.caltech.edu/CaltechETD:etd-12142007-125144

Abstract

Nicotinic acetylcholine receptors (nAChR) are an important family of ligand gated ion channels found throughout the CNS and the PNS. They have been indicated in a series of physiological functions and pathological states. nAChRs have received extensive study in the past as a prototype of the Cys loop LGIC member. Growing interest in developing subtype specific agents targeting nAChRs to treat neurological diseases require more detailed structural and functional information in the numerous members of the nAChR family.

We performed structure-function studies on the chemical scale of several of the most important members of this family using a powerful combination of conventional mutagenesis and unnatural amino acid incorporations. Chapter 2 describes our research in studying the channel gating mechanism of the prototypic nAChR, the muscle type (α₁)₂βγδ. We studied thoroughly the gating interface of the receptor and concluded that the overall charging pattern of the gating interface, and not any specific pairwise electrostatic interactions, controls the gating process in the Cys loop superfamily. Chapter 3 reports our studies in the ligand binding mechanism of the most prevalent neuronal type α4β2 and α7 nAChR. We identified a cation-π interaction and a hydrogen bond employed by nicotine with the α4β2 receptor. These two key interactions are absent or significantly diminished in both the muscle type receptors and in the α7 form of neuronal receptor. In Chapter 4 we studied the ligand binding mechanism of a relatively newly characterized neuronal receptor, α4β4.

From these studies, we found that in the Cys loop superfamily, homology in amino acid sequences and structures do not translate into a shared functional mechanism. In fact, different sets of chemical interactions are adopted between ligands and the receptor, and between amino acids within the ion channel proteins, both in ligand binding and channel gating.

Ion channels are membrane bound multi-subunit macromolecules. We are able to carry out such exhaustive detailed structure-function studies by means of the fast developing methodology of unnatural amino acid incorporation by nonsense suppression. This thesis also describes our effort to improve the efficiency of nonsense suppression. In particular, we designed multiple 21nt small interfering RNA (siRNA) targeting release factor 1 (eRF1) in both HEK cells and Xenopus oocytes, and monitored the nonsense suppression efficiency change in vivo and in vitro by RNA PCR, Western blotting, fluorescence, and electrophysiology (Chapter 5).

Thesis Files