The Tethered Agonist Approach to Mapping Ion-Channel Proteins: Toward a Structural Model for the Agonist-Binding Site of the Nicotinic Acetylcholine Receptor

Citation

Li, Lintong (2003) The Tethered Agonist Approach to Mapping Ion-Channel Proteins: Toward a Structural Model for the Agonist-Binding Site of the Nicotinic Acetylcholine Receptor. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/AMDZ-XE24. https://resolver.caltech.edu/CaltechETD:etd-07312002-120928

Abstract

The integral membrane proteins of neurons and other excitable cells are generally resistant to high-resolution structural tools. In this thesis we present our efforts to probe the structure of the agonist-binding site of the nicotinic acetylcholine receptor (nAChR) using the tethered agonist approach, which combines chemical synthesis, the nonsense suppression methodology for unnatural amino acid incorporation and electrophysiology.

In Chapter 2, we present the results of incorporating a series of tethered quaternary ammonium derivatives of tyrosine into the nAChR using the in vivo nonsense suppression methodology for incorporating unnatural amino acids site-specifically. At three sites, a constitutively active receptor results, but the pattern of activation as a function of chain length is different. At position alpha149, there is a clear preference for a three-carbon tether, while at position alpha93 tethers of 2-5 carbons are comparably effective. At position gamma55/delta57, all tethers except the shortest one can activate the receptor. Based on these and other data, a model for the binding site of the receptor can be developed by analogy to the acetylcholine esterase crystal structure.

In Chapter 3, we report evidence that the N-terminal extracellular domain of nAChR is closely related to acetylcholine binding protein (AChBP), whose crystal structure was solved in May 2001. Based on the model obtained from docking acetylcholine into the structure of AChBP, we designed and incorporated a new tethered agonist, lysyl-carbamylcholine. Incorporation of this tethered agonist at several positions produced constitutively active receptors, with significant activity seen at alpha192, alpha193, and gamma119/delta121. These results demonstrated that the loop E residue gamma119/delta121 on the complementary subunit is very near the agonist-binding site. We also investigated the role of an intersubunit hydrogen bond, which was seen in the crystal structure of AChBP. Incorporation of tryptophan analogs that abolish the hydrogen bonding abilities slowed the desensitization of the receptor, which implied that this hydrogen bond might play a key role in the allosteric transitions of desensitization.

In Chapter 4, we describe our efforts to prepare a short tethered agonist and the results of incorporating it into nAChR at alpha198 by chemical modification of cysteine mutants introduced by nonsense suppression methodology. Methanethiosulphonate ethyltrimethylammonium (MTSET) modification resulted in constitutive activity, which suggested the closeness of alpha198 to the agonist-binding site.

In Chapter 5, methods in molecular biology, electrophysiology and molecular docking, and the synthesis of amino acids and dinucleotide dCA-amino acids are summarized.

Thesis Files