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POSITIVE AND NEGATIVE MODULATORY SITES FOR ETHANOL
IN α1 GLYCINE RECEPTORS
by
Daniel Kenneth Crawford
A Dissertation Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(NEUROSCIENCE)
August 2007
Copyright 2007 Daniel Kenneth Crawford
Object Description
| Title | Positive and negative modulatory sites for ethanol in alpha1 glycine receptors |
| Author | Crawford, Daniel Kenneth |
| Author email | dkcrawfo@usc.edu |
| Degree | Doctor of Philosophy |
| Document type | Dissertation |
| Degree program | Neuroscience |
| School | College of Letters, Arts and Sciences |
| Date defended/completed | 2007-03-27 |
| Date submitted | 2007 |
| Restricted until | Unrestricted |
| Date published | 2007-07-25 |
| Advisor (committee chair) | Alkana, Ronald L. |
| Advisor (committee member) |
Davies, Daryl L. Brinton, Roberta Diaz Byerly, Lou Haworth, Ian |
| Abstract | Ligand-gated ion channels of the cys-loop receptor family have received considerable attention as putative sites of action causing the behavioral effects of ethanol. Studies over the last decade identified several positions in the transmembrane domain critical for ethanol modulation of glycine receptors (GlyRs). Studies to date in the extracellular domain of alpha1GlyRs found that mutations at position 52 in Loop 2 change ethanol sensitivity, alter sensitivity to an ethanol antagonist and can eliminate subunit-dependent differences in ethanol sensitivity between alpha1 and alpha2GlyRs. These findings suggest that the extracellular domain also represents a target for ethanol in GlyRs. We tested this hypothesis by investigating the effect of cysteine substitutions at positions 52 and 267 on responses to n-alcohols and propyl methanethiosulfonate (PMTS) in alpha1GlyRs. We also tested the role of Loop 2 in agonist activation and ethanol modulation by investigating the effect of cysteine point mutations at positions 50-60. Xenopus oocytes expressing human alpha1 wildtype (WT) or mutant GlyRs were voltage-clamped and tested 3-10 days post-injection. In support of the hypothesis, we found that: 1) The A52C mutation changed ethanol sensitivity compared to WT GlyRs; 2) PMTS, a thiol agent held to mimic ethanol action, produced irreversible alcohol-like potentiation in A52C GlyRs; 3) PMTS binding reduced the alcohol cutoff in A52C GlyRs. Further studies used PMTS binding to cysteines to block ethanol action at one site in order to determine its effect at the other site. In these situations, ethanol caused negative modulation when acting at position 52 and positive modulation when acting at position 267.; Latter studies found that cysteine substitutions in Loop 2 produced position-specific changes in glycine sensitivity consistent with beta strand-like structure of the Loop and suggest that odd-numbered positions in Loop 2 interact with other agonist-activation elements. We then found that several Loop 2 cysteine mutations changed ethanol sensitivity and one mutation switched the qualitative ethanol response. Together, these findings support the hypothesis that Loop 2 residues play an important role in agonist activation and ethanol modulation. The findings provide new molecular models and insight into structure-function relationships for GlyRs and possibly other ligand-gated ion channels. |
| Keyword | glycine receptor; ethanol; two-electrode voltage clamp; Xenopus oocyte; molecular model |
| Language | English |
| Part of collection | University of Southern California dissertations and theses |
| Publisher (of the original version) | University of Southern California |
| Place of publication (of the original version) | Los Angeles, California |
| Publisher (of the digital version) | University of Southern California. Libraries |
| Type | texts |
| Legacy record ID | usctheses-m670 |
| Rights | Crawford, Daniel Kenneth |
| Repository name | Libraries, University of Southern California |
| Repository address | Los Angeles, California |
| Repository email | http://www.usc.edu/isd/libraries/services/ask_a_librarian/email/ |
| Filename | etd-Crawford-20070725 |
| Archival file | uscthesesreloadpub_Volume51/etd-Crawford-20070725.pdf |
Description
| Title | Page 1 |
| Full text | POSITIVE AND NEGATIVE MODULATORY SITES FOR ETHANOL IN α1 GLYCINE RECEPTORS by Daniel Kenneth Crawford A Dissertation Presented to the FACULTY OF THE GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY (NEUROSCIENCE) August 2007 Copyright 2007 Daniel Kenneth Crawford |
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