The long term goal in this project remains the elucidation of the structure and function of nicotinic acetylcholine receptors (nAChRs). Classical approaches to discern functionally significant neuronal nAChR subtypes in the central nervous system (CNS) have been frustrated by the limited number of selective pharmacological agents. Structure-based information will be used in this project to drive the development of new research tools to investigate nAChR subunit-specific functionality in the nervous system.
The first aim focuses on the characterization of a """"""""Knock-In"""""""" mouse, Chrna3tm1(Hwrt), created through homologous recombination-mediated targeted gene replacement. Targeted DMAencoding five muscle-type a1-derived amino acid substitutions confers functional sensitivity of receptors to nanomolar a-bungarotoxin (Bgtx) even in those cases where only 1 of the 2 receptor a3 subunits is mutant. Heterozygous mice express hybrid nAChRs containing one mutant and one wild-type a3 subunit, but are otherwise normal phenotypically. Consistent with a stochastic expression of hybrid receptors, -2/3 of the nicotinic response in sympathetic neurons from Met mice can be blocked by Bgtx. Biochemical and electrophysiological methods will be used to fully assess the functional consequences of this mutation in heterozygous mice backcrossed into the C57BI/6/J background. The expression of the mutant a3 subunit in the CNS will be investigated by fluorescence, autoradiography, and micro-injection of Bgtx into discrete brain regions rich in a3. In the second aim, Bgtx-sensitive P2, (33,04 and a5 subunits will be prepared and characterized electro- physiologically following heterologous expression in oocytes and in adenovirus-transfected neurons. These results will determine the future feasibility of generating Bgtx-sensitive knock-in mice in these 4 subunits. Relevance: Nicotine is an extremely addictive drug responsible for up to 20% of all preventable mortality in the western world. It also significantly enhances cognitive performance, and some inherited forms of epilepsy involve nicotinic receptors. Loss of cholinergic neurons is implicated in Alzheimer's disease, a disorder with no effective treatment. Understanding the functional role of nicotinic receptors in the CNS therefore has significant potential to benefit human health. In addition, the results from this study could lead to the development of therapeutic drugs reproducing some of the beneficial effects of nicotine.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
3R01GM032629-22S1
Application #
7937425
Study Section
Special Emphasis Panel (ZRG1-MDCN-F (02))
Program Officer
Dunsmore, Sarah
Project Start
2009-09-30
Project End
2011-05-31
Budget Start
2009-09-30
Budget End
2011-05-31
Support Year
22
Fiscal Year
2009
Total Cost
$125,431
Indirect Cost
Name
Brown University
Department
Pharmacology
Type
Schools of Medicine
DUNS #
001785542
City
Providence
State
RI
Country
United States
Zip Code
02912
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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 :
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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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