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Binding Site Structure and Stoichiometry in Serotonin Type 3 Receptors


Miles, Timothy Francis (2015) Binding Site Structure and Stoichiometry in Serotonin Type 3 Receptors. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/Z9T151MC .


This dissertation primarily describes studies of serotonin type 3 (5-HT3) receptors of the Cys-loop super-family of ligand gated ion channels. The first chapter provides a general introduction to these important proteins and the methods used to interrogate their structure and function. The second chapter details the delineation of a structural unit of the ligand binding site of homomeric 5-HT3A receptors on which the ligands serotonin (5-HT) and m-chlorophenyl biguanide (mCPBG) are reliant for effective receptor activation. Unnatural amino acid mutagenesis results show that the details of each ligand’s interaction with this organizing feature of the binding site differ, providing insights into general principles of receptor activation.

The third chapter describes a study in which florescent protein fusions of the A and B subunits of the heteromeric 5-HT3AB receptor are employed to determine the subunit stoichiometry and order within functional receptors. Strong evidence is found for an A3B2 stoichiometry with A-A-B-A-B order. The fourth chapter investigates the potential for ligand binding across heteromeric binding sites in the 5-HT3AB receptor. Unlike serotonin, mCPBG is found to bind the receptor at heteromeric binding sites. Further mCPBG is capable of allosterically modulating the response of serotonin on the 5-HT3AB receptor from these heteromeric sites.

Finally, the fifth chapter describes progress towards the application of unnatural amino acid mutagenesis to an important new class of proteins, transcription factors. Experiments optimizing novel methods for the detection of function are described, using RARα of the nuclear receptor family of transcription factors.

Item Type:Thesis (Dissertation (Ph.D.))
Subject Keywords:Cys-loop receptor, unnatural amino acid mutagenesis, serotonin, electrophysiology
Degree Grantor:California Institute of Technology
Division:Biology and Biological Engineering
Major Option:Molecular Biology and Biochemistry
Thesis Availability:Public (worldwide access)
Research Advisor(s):
  • Dougherty, Dennis A.
Thesis Committee:
  • Lester, Henry A. (chair)
  • Deshaies, Raymond Joseph
  • Rees, Douglas C.
  • Dougherty, Dennis A.
Defense Date:11 July 2014
Record Number:CaltechTHESIS:07142014-132743727
Persistent URL:
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:8537
Deposited By: Timothy Miles
Deposited On:30 Jun 2015 23:24
Last Modified:06 Nov 2019 17:45

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