We wish to understand how the nicotinic acetylcholine receptor (AChR) functions as a ligand-gated ion channel and to understand how drugs and toxins interact with the AChR to alter its function. Torpedo electric tissue will be fractionated to. isolate postsynaptic membranes to be used in biochemical studies designed to: 1) identify functional domains (ligand binding sites, ion channel) in terms of the known amino acid sequences of AChR subunits and to identify differences in structure of those domains that distinguish between the binding of agonists and antagonists or between different conformational states of the AChR, and 2) provide a general description of the three dimensional structure of the AChR in terms of the regions of each subunit exposed at extracellular or cytoplasmic surfaces, at subunit interfaces, or in contact with lipid. Radiolabeled affinity labels and structural probes will be covalently incorporated into AChR, and labeled AChR subunits will be isolated and degraded so that sites of labeling will be determined by N-terminal sequence analysis of isolated labeled peptides. The structural studies will provide a definition of particular amino acids and regions contained within binding sites, at subunit interfaces, or at the protein- lipid interface, but they do not of their own assess the importance of the identified amino acids as determinants of ligand binding affinity or their involvement in the mechanism of channel gating. To address these issues a third research goal is to test models of AChR structure derived from the structural studies by analysis of functional properties of mutant AChRs expressed in Xenopus oocytes. The equilibrium binding affinity of agonists and antagonists will be assessed by radioligand binding assays, while AChR function will be assessed by electrophysiological techniques. Point mutations will be introduced to change amino acids predicted to be important for ligand binding affinity or for channel-gating, and additional mutations will be made of amino acids predicted to be important in the propagation of structural changes from the ACh binding site to the ion channel.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS019522-13
Application #
2263610
Study Section
Neurological Sciences Subcommittee 1 (NLS)
Project Start
1982-09-01
Project End
1999-11-30
Budget Start
1993-12-01
Budget End
1994-11-30
Support Year
13
Fiscal Year
1994
Total Cost
Indirect Cost
Name
Harvard University
Department
Biology
Type
Schools of Medicine
DUNS #
082359691
City
Boston
State
MA
Country
United States
Zip Code
02115
Kasheverov, Igor E; Chiara, David C; Zhmak, Maxim N et al. (2006) alpha-Conotoxin GI benzoylphenylalanine derivatives. (1)H-NMR structures and photoaffinity labeling of the Torpedo californica nicotinic acetylcholine receptor. FEBS J 273:1373-88
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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
Chiara, David C; Trinidad, Jonathan C; Wang, Dong et al. (2003) Identification of amino acids in the nicotinic acetylcholine receptor agonist binding site and ion channel photolabeled by 4-[(3-trifluoromethyl)-3H-diazirin-3-yl]benzoylcholine, a novel photoaffinity antagonist. Biochemistry 42:271-83
Sullivan, Deirdre; Chiara, David C; Cohen, Jonathan B (2002) Mapping the agonist binding site of the nicotinic acetylcholine receptor by cysteine scanning mutagenesis: antagonist footprint and secondary structure prediction. Mol Pharmacol 61:463-72
Ramarao, M K; Bianchetta, M J; Lanken, J et al. (2001) Role of rapsyn tetratricopeptide repeat and coiled-coil domains in self-association and nicotinic acetylcholine receptor clustering. J Biol Chem 276:7475-83
Xie, Y; Cohen, J B (2001) Contributions of Torpedo nicotinic acetylcholine receptor gamma Trp-55 and delta Trp-57 to agonist and competitive antagonist function. J Biol Chem 276:2417-26
Gallagher, M J; Chiara, D C; Cohen, J B (2001) Interactions between 3-(Trifluoromethyl)-3-(m-[(125)I]iodophenyl)diazirine and tetracaine, phencyclidine, or histrionicotoxin in the Torpedo series nicotinic acetylcholine receptor ion channel. Mol Pharmacol 59:1514-22
Blanton, M P; Lala, A K; Cohen, J B (2001) Identification and characterization of membrane-associated polypeptides in Torpedo nicotinic acetylcholine receptor-rich membranes by hydrophobic photolabeling. Biochim Biophys Acta 1512:215-24

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