The nicotinic acetylcholine receptor (AChR) is a multimeric, integral membrane glycoprotein that functions as a ligand gated channel at the neuromuscular junction. The biosynthesis, assembly, membrane insertion and localization of the AChR complex provides an attractive system for the study of the mechanisms underlying these important cellular functions. Three complementary approaches will be used to provide additional fundamental information concerning AChR biogenesis, regulation, structure and function. 1) We plan to extend our monoclonal antibody (mAb) studies to focus on the extracellular domains of mouse muscle AChR including the ligand binding site and to identify the characteristics of cross-reacting antigens observed in a variety of electrically excitable tissues, including Drosophila central nervous system (CNS), guinea pig ileum smooth muscle, rat sympathetic ganglia, and the rat pheochromocytoma PC12 cell line. 2) Somatic cell genetic techniques will be applied to the questions of AChR regulation and structure-function relationship. Fluorescence-activated cell sorting in combination with specific immunotoxins and replica-plating will be used to obtain muscle cell variants with altered regulatory, biosynthetic, or structural features involving the AChR. 3) The protein-blotting approach will be used to provide additional structural information on the AChR as well as on possible evolutionarily related structures such as the Alpha-bungarotoxin (BuTX) binding site in the CNS of lower vertebrates. We plan also to utilize this approach to identify the amino acid sequences making up the epitopes responsible for various cross-reactions and to thus map the immunologically identifiable sites with respect to other structural features of the AChR.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM032629-04
Application #
3281656
Study Section
Neurological Sciences Subcommittee 1 (NLS)
Project Start
1983-04-01
Project End
1990-03-31
Budget Start
1986-04-01
Budget End
1987-03-31
Support Year
4
Fiscal Year
1986
Total Cost
Indirect Cost
Name
Yale University
Department
Type
Schools of Medicine
DUNS #
082359691
City
New Haven
State
CT
Country
United States
Zip Code
06520
Peng, Can; Chen, Weihua; Sanders, Tanya et al. (2010) Chemical synthesis and characterization of two ýý4/7-conotoxins. Acta Biochim Biophys Sin (Shanghai) 42:745-53
Peng, Can; Ye, Mingyu; Wang, Yanfang et al. (2010) A new subfamily of conotoxins belonging to the A-superfamily. Peptides 31:2009-16
Moise, Leonard; Liu, Jing; Pryazhnikov, Evgeny et al. (2010) K(V)4.2 channels tagged in the S1-S2 loop for alpha-bungarotoxin binding provide a new tool for studies of channel expression and localization. Channels (Austin) 4:115-23
Paulo, Joao; Brucker, William; Hawrot, Edward (2009) Proteomic Analysis of an 7 Nicotinic Acetylcholine Receptor Interactome. J Proteome Res :
Paulo, Joao A; Hawrot, Edward (2009) Effect of homologous serotonin receptor loop substitutions on the heterologous expression in Pichia of a chimeric acetylcholine-binding protein with alpha-bungarotoxin-binding activity. Protein Expr Purif 67:76-81
Paulo, Joao A; Hawrot, Edward (2009) A radioisotope label-free alpha-bungarotoxin-binding assay using BIAcore sensor chip technology for real-time analysis. Anal Biochem 389:86-8
Caffery, Philip M; Krishnaswamy, Arjun; Sanders, Tanya et al. (2009) Engineering neuronal nicotinic acetylcholine receptors with functional sensitivity to alpha-bungarotoxin: a novel alpha3-knock-in mouse. Eur J Neurosci 30:2064-76
Peng, Can; Chen, Weihua; Han, Yuhong et al. (2009) Characterization of a novel alpha4/4-conotoxin, Qc1.2, from vermivorous Conus quercinus. Acta Biochim Biophys Sin (Shanghai) 41:858-64
Liu, Li; Chew, Geoffrey; Hawrot, Edward et al. (2007) Two potent alpha3/5 conotoxins from piscivorous Conus achatinus. Acta Biochim Biophys Sin (Shanghai) 39:438-44
Sanders, Tanya; Hawrot, Edward (2004) A novel pharmatope tag inserted into the beta4 subunit confers allosteric modulation to neuronal nicotinic receptors. J Biol Chem 279:51460-5

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