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Single-molecule studies of ion channels expressing unnatural amino acids

Citation

Shanata, Jai Anand Pattur (2011) Single-molecule studies of ion channels expressing unnatural amino acids. Dissertation (Ph.D.), California Institute of Technology. http://resolver.caltech.edu/CaltechTHESIS:06082011-080856211

Abstract

The nicotinic acetylcholine receptors are pentameric ligand-gated ion channels that mediate fast synaptic transmission in the brain and peripheral nervous system. After an introduction (Chapter 1), Chapter 2 describes my development of techniques to combine single-channel and whole-cell recording with nonsense suppression. Having established the feasibility of the combined use of single-channel and whole-cell recording, in Chapter 3 we developed a method to identify the functional interactions of amino acids that are physically far apart in a protein. This is fundamentally a whole-cell recording method to find allosteric interactions in ion channels. The significance of this method is strongly supported by single-channel measurements. Additionally, the relationship between the single-channel gating equilibrium constant, theta, and the whole-cell measurement of EC50 is considered.

In Chapter 4, I describe my progress towards measuring the channel opening rate of the fetal and adult muscle-type nicotinic acetylcholine receptors. Multiple different agonists are used, including acetylcholine, choline, and tetramethylammonium. Single-channel data are reported for the wild-type receptors as well as for receptors with the unnatural amino acid 5-F-Trp (monofluoro-Trp). Data are reported for multiple concentrations for a mutated fetal nAChR, and QuB is used to fit various possible models and estimate theta for this mutant.

A major aim of this dissertation was to use single-molecule studies of ion channels expressing unnatural amino acids to provide even more convincing evidence for cation-pi interactions at the binding sites of ligand-gated ion channels, specifically the neuronal nicotinic acetylcholine receptor. Chapter 5 describes the combined application of single-channel, whole-cell, and unnatural amino acid mutagenesis to the specific question of how two molecules—nicotine and Chantix® (varenicline)—bind to the alpha4beta2 brain receptor. In Chapter 6, I describe single-channel experiments that establish a method for distinguishing between the two known stoichiometries of the wild type alpha4beta2 brain receptor. Specifically, I identify a difference in the rectification properties of the high and low affinity receptors.

Item Type:Thesis (Dissertation (Ph.D.))
Subject Keywords:ion channels, single-channel recording, organic chemistry, receptors, unnatural amino acids, single-molecule, acetylcholine, choline, nicotine, varenicline, cation-pi interaction, molecular recognition, kinetics
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)
  • Rees, Douglas C.
  • Lester, Henry A.
  • Clemons, William M.
  • Dougherty, Dennis A.
Defense Date:1 June 2011
Author Email:jshanata (AT) gmail.com
Funders:
Funding AgencyGrant Number
NIH34407
NIH11756
NRSA Training GrantUNSPECIFIED
Tobacco-Related Disease Research Program of the University of California16RT-0160
Record Number:CaltechTHESIS:06082011-080856211
Persistent URL:http://resolver.caltech.edu/CaltechTHESIS:06082011-080856211
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:6509
Collection:CaltechTHESIS
Deposited By: Jai Shanata
Deposited On:14 Jun 2011 22:10
Last Modified:07 Feb 2014 18:33

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