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Chemical-scale studies of the nicotinic and muscarinic acetylcholine receptors

Citation

Torrice, Michael McCann (2009) Chemical-scale studies of the nicotinic and muscarinic acetylcholine receptors. Dissertation (Ph.D.), California Institute of Technology. http://resolver.caltech.edu/CaltechETD:etd-08072008-103726

Abstract

This dissertation describes three chemical-scale studies of neuroreceptor structure and function. Incorporation of unnatural amino acids into two acetylcholine receptors—the nicotinic acetylcholine receptor (nAChR) and the M2 muscarinic acetylcholine receptor (M2AChR)—and an electrophysiology assay of receptor function were performed in each of the studies. The nAChR is a ligand-gated ion channel (LGIC) and the M2AChR is a G-protein-coupled receptor (GPCR).

In Chapter 2, a highly conserved aspartate residue (D89) that is near the agonist binding site of the nAChR was probed for its role in agonist binding. We found that the side chain of D89 establishes a redundant network of hydrogen bonds and preorganizes the agonist binding site by positioning a critical agonist-binding residue, tryptophan 149 (W149). Previous studies of a D89N mutant led to the proposal that a negative charge at D89 was essential for receptor function. However, our studies show that neutral side chains at position 89 function well, only if an unfavorable electrostatic clash is avoided.

Chapter 3 describes our attempts to incorporate unnatural amino acids into the M2AChR, a GPCR. GPCR activity is assayed through second messenger signaling pathways, unlike the direct readout assays of LGICs. These second messenger pathways require significant amounts of optimization to create assays that produce reliable and robust data. In our experiments, variability of dose-response relationship data between batches of cells was the most significant concern. Several factors were investigated to reduce this batch-to-batch variability. After a reliable means to assay M2AChR function was found, we performed a preliminary search for tryptophan residues in the agonist binding site that form a cation-pi interaction with acetylcholine.

Finally, in Chapter 4, we discuss the use of hydroxy acids to scan the alpha-M1 transmembrane helix of the nAChR for residues that undergo structural rearrangements during gating. Hydroxy acids disrupt hydrogen bonding in protein backbones and thus provide a means to detect backbone interactions that form or break during gating. The hydroxy acid analog of valine, valic acid (Vah), was incorporated at ten positions along the alpha-M1 helix. Backbone mutations at five residues on the intracellular side of a conserved proline (P221) produced shifts in dose-response relationships.

Item Type:Thesis (Dissertation (Ph.D.))
Subject Keywords:G-protein-coupled receptors; ligand-gated ion channels; nitro amino acids; unnatural amino acids
Degree Grantor:California Institute of Technology
Division:Chemistry and Chemical Engineering
Major Option:Chemistry
Thesis Availability:Public (worldwide access)
Research Advisor(s):
  • Dougherty, Dennis A.
Thesis Committee:
  • Dervan, Peter B. (chair)
  • Tirrell, David A.
  • Dougherty, Dennis A.
  • Goddard, William A., III
Defense Date:25 July 2008
Author Email:torrice (AT) gmail.com
Record Number:CaltechETD:etd-08072008-103726
Persistent URL:http://resolver.caltech.edu/CaltechETD:etd-08072008-103726
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:3041
Collection:CaltechTHESIS
Deposited By: Imported from ETD-db
Deposited On:11 Nov 2008
Last Modified:26 Dec 2012 02:56

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