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Type of Document	Dissertation
Author	Beene, Darren Lee
Author's Email Address	beened@ohsu.edu
URN	etd-04262005-153907
Persistent URL	http://resolver.caltech.edu/CaltechETD:etd-04262005-153907
Title	Chemical scale investigations of ligand-gated ion channels using unnatural amino acids
Degree	PhD
Option	Chemistry
Advisory Committee	Advisor Name Title Peter B. Dervan Committee Chair Dennis A. Dougherty Committee Member Richard W. Roberts Committee Member Robert H. Grubbs Committee Member
Keywords	nonsense suppression proline isomerization cation-pi interaction serotonin 5-hydroxytryptamine acetylcholine nicotine M2-M3 loop GABA
Date of Defense	2004-12-09
Availability	unrestricted
Abstract The Cys loop receptors, a family of ligand-gated ion channels, mediate fast synaptic transmission throughout the peripheral and central nervous systems. These are large multisubunit proteins, whose primary function is to transduce a chemical signal, binding of a neurotransmitter, into an electrical signal, ion flux across the cell membrane. These receptors have been implicated in several disease states and represent major therapeutic targets. The work presented in this thesis focuses on the chemical-scale elucidation of Cys loop receptors. The main approach of this work is the structure-function study using in vivo nonsense suppression methods. This technique allows for the site-specific incorporation of an unnatural amino acid into a protein expressed in a living cell. Nonsense suppression methods were used to incorporate a series of fluorinated tryptophan derivatives into the binding site of the 5-HT3R. This study identified a cation-pi interaction between Trp 183 and the neurotransmitter, serotonin. A similar study using fluorinated phenylalanine derivatives identified a cation-pi binding site at Tyr 198 in the GABAC receptor. These studies build on previous work from our research group and provide further evidence that the cation-pi interaction is a common feature in ligand recognition by Cys loop receptors. Nonsense suppression was also used to examine the role of several tyrosine residues in the 5-HT3R. Here the findings demonstrated that the side chains of Tyr 143 and 153 make functionally important hydrogen bonds. These data were used to refine several computational models of serotonin docked into the binding site. Structure-function studies of two conserved prolines in the M2-M3 loop showed that this region of the receptor is involved in the conformational changes associated with receptor activation. The data also provide preliminary evidence that Pro 308 may serve as hinge for the gating movement of the M2 helix.
Files	Filename       Size       Approximate Download Time (Hours:Minutes:Seconds)     28.8 Modem   56K Modem   ISDN (64 Kb)   ISDN (128 Kb)   Higher-speed Access    DLB_Chap1.pdf 805.93 Kb 00:03:43 00:01:55 00:01:40 00:00:50 00:00:04   DLB_Chap2.pdf 1.47 Mb 00:06:49 00:03:30 00:03:04 00:01:32 00:00:07   DLB_Chap3.pdf 1.02 Mb 00:04:44 00:02:26 00:02:08 00:01:04 00:00:05   DLB_Chap4.pdf 2.08 Mb 00:09:36 00:04:56 00:04:19 00:02:09 00:00:11   DLB_Chap5.pdf 997.30 Kb 00:04:37 00:02:22 00:02:04 00:01:02 00:00:05   DLB_Thesis.pdf 6.08 Mb 00:28:08 00:14:28 00:12:39 00:06:19 00:00:32   DLB_Title_pages.pdf 355.77 Kb 00:01:38 00:00:50 00:00:44 00:00:22 00:00:01