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Nicotine Normalizes Intracellular Subunit Stoichiometry of Nicotinic Receptors Carrying Mutations Linked to Autosomal Dominant Nocturnal Frontal Lobe Epilepsy

Son, Cagdas D. and Moss, Fraser J. and Cohen, Bruce N. and Lester, Henry A. (2009) Nicotine Normalizes Intracellular Subunit Stoichiometry of Nicotinic Receptors Carrying Mutations Linked to Autosomal Dominant Nocturnal Frontal Lobe Epilepsy. Molecular Pharmacology, 75 (5). pp. 1137-1148. ISSN 0026-895X. PMCID PMC2672806. doi:10.1124/mol.108.054494.

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Autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) is linked with high penetrance to several distinct nicotinic receptor (nAChR) mutations. We studied (α4)_3(2β)_2 versus (α4)_2(β2)_3 subunit stoichiometry for five channel-lining M2 domain mutations: S247F, S252L, 776ins3 in α4, V287L, and V287M in β2. α4 and β2 subunits were constructed with all possible combinations of mutant and wild-type (WT) M2 regions, of cyan and yellow fluorescent protein, and of fluorescent and nonfluorescent M3-M4 loops. Sixteen fluorescent subunit combinations were expressed in N2a cells. Forster resonance energy transfer (FRET) was analyzed by donor recovery after acceptor photobleaching and by pixel-by-pixel sensitized emission, with confirmation by fluorescence intensity ratios. Because FRET efficiency is much greater for adjacent than for nonadjacent subunits and the α4 and β2 subunits occupy specific positions in nAChR pentamers, observed FRET efficiencies from (α4)_3(β2)_2 carrying fluorescent α4 subunits were significantly higher than for (α4)_2(β2)_3; the converse was found for fluorescent 2 subunits. All tested ADNFLE mutants produced 10 to 20% increments in the percentage of intracellular (α4)_3(β2)_2 receptors compared with WT subunits. In contrast, 24- to 48-h nicotine (1 µM) exposure increased the proportion of (α4)_2(β2)_3 in WT receptors and also returned subunit stoichiometry to WT levels for α4S248F and β2V287L nAChRs. These observations may be relevant to the decreased seizure frequency in patients with ADNFLE who use tobacco products or nicotine patches. Fluorescence-based investigations of nAChR subunit stoichiometry may provide efficient drug discovery methods for nicotine addiction or for other disorders that result from dysregulated nAChRs.

Item Type:Article
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URLURL TypeDescription CentralArticle
Moss, Fraser J.0000-0002-8519-6991
Lester, Henry A.0000-0002-5470-5255
Additional Information:© 2009 by the American Society for Pharmacology and Experimental Therapeutics. Received January 2, 2009; accepted February 23, 2009. This work was supported by the National Institutes of Health National Institute of Neurological Disorders and Stroke [Grant NS11756]; by Targacept, Inc.; by a fellowship from Philip Morris USA/International (to C.D.G.); and by a fellowship from the American Heart Association (to F.J.M.). We thank Princess Imoukhuede, Rigo Pantoja, Rahul Srinivasan, Larry Wade, and Ben Corry (University of Western Australia) for discussion and Jeff Larsen (Nikon) for much technical help.
Funding AgencyGrant Number
American Heart AssociationUNSPECIFIED
Issue or Number:5
PubMed Central ID:PMC2672806
Record Number:CaltechAUTHORS:20090914-084414850
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Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:15826
Deposited By: Tony Diaz
Deposited On:16 Sep 2009 23:23
Last Modified:08 Nov 2021 23:22

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