""""""""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.
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