Investigations of Ion Channel Structure and Function. I. Studies of Nicotine Binding to the Acetylcholine Receptor. II. Development of Tools for Studying Learning and Memory with Unnatural Amino Acids

Citation

Petersson, Ernest James (2005) Investigations of Ion Channel Structure and Function. I. Studies of Nicotine Binding to the Acetylcholine Receptor. II. Development of Tools for Studying Learning and Memory with Unnatural Amino Acids. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/yhty-re87. https://resolver.caltech.edu/CaltechETD:etd-05242005-172213

Abstract

This dissertation can be divided into two main sections:

I. In previous studies, we have used fluorinated tryptophan derivatives to conclusively identify a cation-pi interaction with Trp 149 in the binding of acetylcholine (ACh) to the muscle-type nicotinic acetylcholine receptor (nAChR). We have incorporated mimics of ACh, termed tethered agonists, in the binding site to produce self-activating channels. Using tertiary tethered agonists that would only become cations and activate the channel when protonated, we identified a perturbed pKa for the binding pocket, which has implications for the binding of tertiary agonists like nicotine (Nic). It has been shown that Nic does not participate in a straight-forward cation-pi interaction as ACh does. We have examined a hydrogen bond between the Nic pyrrolidine N-H and the backbone carbonyl of Trp149 by introducing an ester linkage at this point, weakening the carbonyl H-bond accepting ability. Calculations performed on hydrogen bound complexes of ACh, Nic, and the Nic analog epibatidine (Epi) explain the trends observed for ligand activation of the nAChR. Expanding upon this study, we have performed molecular dynamics (MD) simulations of models of the ligand binding domain of the nAChR. Ligand-bound structures from these simulations have been taken on to quantum mechanical/molecular mechanical (QMMM) calculations to model the effects of unnatural amino acid mutations in an environment that simulates the full nAChR binding pocket.

II. The nAChR is essential to neurotransmission at the junction between nerve and muscle cells, and it plays an important role in many central nervous system processes. However, its role in learning and memory is limited, at least in our current molecular models of these events. In a sense, the formation of a memory consists of the strengthening of some synaptic connections and the weakening of others. These processes, termed long term potentiation (LTP) and depression (LTD) respectively, are primarily governed by modifications to glutamate receptors (GluRs). We have developed tools for studying the mechanism and timecourse of these modifications (of phosphorylation in particular), and we have demonstrated the first incorporation of unnatural amino acids into a GluR.

Thesis Files