""""""""Structural Analysis of Acetylcholine and GABA alpha Receptors"""""""" The broad, long term objectives of this proposal are to elucidate the mechanisms which underlie the functioning of the nicotinic acetylcholine receptor (AChR) family of ligand-gated ion channels (LGIC). Each of these receptors are important in mediating synaptic transmission in the nervous systems and constitute important target sites of many therapeutic drugs. A critical step in determining how these receptors function in identifying and examining in detail the structural elements which mediate receptor activity.
The specific aims of this project are: 1) To determine the secondary structure of the transmembrane segments which comprise the ion channel, 2) Map out the topology of the agonist-binding site and or the transmembrane segments, and 3) Probe the structure of the type a gamma-aminobutyric acid (GABA alpha) receptor ion channel. Research design and methods: In studies using AChRs isolated from Torpedo electric organ, proteolytic fragments containing one or more membrane-spanning segments (M1-M4) will be isolated and the secondary structure examined using both circular dichroic (CD) and fourier transform infrared (FTIR) spectroscopy. The topology of the agonist-binding site and transmembrane segments will be assessed using several different site-selective radiolabeled probes. Labeled sites will be determined by N-terminal sequence analysis of isolated proteolytic fragments. Using GABA alpha receptors affinity purified from bovine cerebral cortex, the structure of the channel will be probed using 3- trifluoromethyl-3-(m-[125I]iodophenyl) diazirine ([125I]TID), a small uncharged photoreactive ligand and potent noncompetitive antagonist of the AChR. The sites of specific [125I]TID incorporation will be determined by sequencing of isolated proteolytic fragments and compared to results obtained with the AChR.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
First Independent Research Support & Transition (FIRST) Awards (R29)
Project #
5R29NS035786-04
Application #
6187353
Study Section
Neurological Sciences Subcommittee 1 (NLS)
Program Officer
Talley, Edmund M
Project Start
1997-08-01
Project End
2002-04-30
Budget Start
2000-05-01
Budget End
2001-04-30
Support Year
4
Fiscal Year
2000
Total Cost
$105,653
Indirect Cost
Name
Texas Tech University
Department
Pharmacology
Type
Schools of Medicine
DUNS #
609980727
City
Lubbock
State
TX
Country
United States
Zip Code
79430
Hamouda, Ayman K; Chiara, David C; Sauls, Daniel et al. (2006) Cholesterol interacts with transmembrane alpha-helices M1, M3, and M4 of the Torpedo nicotinic acetylcholine receptor: photolabeling studies using [3H]Azicholesterol. Biochemistry 45:976-86
Arias, Hugo R; Trudell, James R; Bayer, Erin Z et al. (2003) Noncompetitive antagonist binding sites in the torpedo nicotinic acetylcholine receptor ion channel. Structure-activity relationship studies using adamantane derivatives. Biochemistry 42:7358-70
Arias, Hugo R; Blanton, Michael P (2002) Molecular and physicochemical aspects of local anesthetics acting on nicotinic acetylcholine receptor-containing membranes. Mini Rev Med Chem 2:385-410
Arias, Hugo R; McCardy, Elizabeth A; Bayer, Erin Z et al. (2002) Allosterically linked noncompetitive antagonist binding sites in the resting nicotinic acetylcholine receptor ion channel. Arch Biochem Biophys 403:121-31
Arias, H R; McCardy, E A; Gallagher, M J et al. (2001) Interaction of barbiturate analogs with the Torpedo californica nicotinic acetylcholine receptor ion channel. Mol Pharmacol 60:497-506
Arias, H R; McCardy, E A; Blanton, M P (2001) Characterization of the dizocilpine binding site on the nicotinic acetylcholine receptor. Mol Pharmacol 59:1051-60
Blanton, M P; McCardy, E A; Fryer, J D et al. (2000) 5-hydroxytryptamine interaction with the nicotinic acetylcholine receptor. Eur J Pharmacol 389:155-63
Barrantes, F J; Antollini, S S; Blanton, M P et al. (2000) Topography of nicotinic acetylcholine receptor membrane-embedded domains. J Biol Chem 275:37333-9
Blanton, M P; McCardy, E A; Gallagher, M J (2000) Examining the noncompetitive antagonist-binding site in the ion channel of the nicotinic acetylcholine receptor in the resting state. J Biol Chem 275:3469-78
Arias, H R; Blanton, M P (2000) Alpha-conotoxins. Int J Biochem Cell Biol 32:1017-28

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