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Chemical scale investigations of the gating mechanism of ion channels

Citation

Lee, Lori WaiHang (2007) Chemical scale investigations of the gating mechanism of ion channels. Dissertation (Ph.D.), California Institute of Technology. http://resolver.caltech.edu/CaltechETD:etd-01152007-080704

Abstract

The studies contained in this dissertation are aimed at utilizing chemistry to understand neurobiology and neuronal communication. Chapters 2 and 3 both address the gating of ion channels, describing structure-function studies to shed light on the gating mechanisms of two classes of ion channels. Chapter 2 studies the gating mechanism of the mechanosensitive channel of small conductance (MscS), which is voltage modulated. Elucidating the mechanism of voltage sensation in MscS may provide insight into how voltage-gated channels translate a change in membrane potential to channel gating. The research discussed in Chapter 2 is aimed at elucidating the role of two arginine residues, in the TM1 and TM2 of MscS, in voltage sensing. We generated two MscS mutants, Arg46Ala and Arg74Ala, to evaluate the effects of "neutralizing" the charged side chain on the voltage sensing ability of the channel. The mutants were evaluated using single channel analysis in E. coli spheroplasts. Our preliminary results indicated a potentially significant role for Arg46 in the voltage sensitivity of MscS, however this data set is not extensive due to inconsistency in the spheroplasts preparation.

In Chapter 3, we utilized nonsense suppression to incorporate unnatural amino acids to study the gating of the cation-selective Cys-loop family of ion channel receptors. Specifically, it describes work aimed at elucidating the role of cis-trans isomerization of a proline residue in the gating mechanism of the serotonin-gated 5-hydroxy-tryptamine receptor 3A (5-HT3A) and the nicotinic acetylcholine receptor. A series of proline analgues, of varying cis preference were incorporated at proline 308 in the M2-M3 loop of the 5-HT3A receptor using in vivo nonsense suppression methodology in a Xenopus oocyte expression system. Electrophysiological analysis of the mutant channels revealed a linear relationship between the cis preference of the proline analog and the EC50 of the mutant channel—suggesting that proline 308 may serve as a hinge during the gating 5-HT3A. From these data, we proposed a model of gating for the 5-HT3A receptor. Initial results from similar studies in nAChR suggests that the analogous proline does not play a role in its gating.

Lastly, Chapter 4 addresses the role of fucose-galactose carbohydrates in learning and memory. It aims to identify lectins to fucose-alpha(1-2)-galactose as well as identify the corresponding glycoproteins bearing fucose-alpha(1-2)-galactose. Chemical probes were synthesized and used to study fucose-alpha(1-2)-galactose binding proteins. One of the probes was used to demonstrate the existence of fucose-alpha(1-2)-galactose binding proteins in hippocampal neurons. Furthermore, initial results from experiments with a photoreactive probe suggested that the design of our probe is sufficient to isolate fucose-alpha(1-2)-galactose binding proteins from the brain. Additionally, we were able to use antibodies specific to fucose-alpha(1-2)-galactose epitopes to examine fucose-alpha(1-2)-galactose bearing glycoproteins in the brain. Overall, results from both studies utilizing chemical probes and molecular probes strongly suggest that the modifications of proteins with fucose-alpha(1-2)-galactose epitopes and the expression of fucose-alpha(1-2)-galactose binding proteins are developmentally regulated.

Item Type:Thesis (Dissertation (Ph.D.))
Subject Keywords:5-hydroxytryptamine; arginine; fucose-galactose; M2-M3 loop; MscS; nicotine; nonsense suppression; proline isomerization; voltage-gated
Degree Grantor:California Institute of Technology
Division:Chemistry and Chemical Engineering
Major Option:Chemistry
Thesis Availability:Restricted to Caltech community only
Research Advisor(s):
  • Dougherty, Dennis A.
Thesis Committee:
  • Dougherty, Dennis A. (chair)
  • Rees, Douglas C.
  • Lester, Henry A.
  • Dervan, Peter B.
  • Goddard, William A., III
Defense Date:18 December 2006
Record Number:CaltechETD:etd-01152007-080704
Persistent URL:http://resolver.caltech.edu/CaltechETD:etd-01152007-080704
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:190
Collection:CaltechTHESIS
Deposited By: Imported from ETD-db
Deposited On:08 Mar 2007
Last Modified:26 Dec 2012 02:27

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