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Subcellular Trafficking, Pentameric Assembly, and Subunit Stoichiometry of Neuronal Nicotinic Acetylcholine Receptors Containing Fluorescently Labeled α6 and β3 Subunits

Drenan, Ryan M. and Nashmi, Raad and Imoukhuede, Princess and Just, Herwig and McKinney, Sheri and Lester, Henry A. (2008) Subcellular Trafficking, Pentameric Assembly, and Subunit Stoichiometry of Neuronal Nicotinic Acetylcholine Receptors Containing Fluorescently Labeled α6 and β3 Subunits. Molecular Pharmacology, 73 (1). pp. 27-41. ISSN 0026-895X. doi:10.1124/mol.107.039180. https://resolver.caltech.edu/CaltechAUTHORS:20200422-074016834

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Abstract

Neuronal nicotinic acetylcholine (ACh) receptors are ligand-gated, cation-selective ion channels. Nicotinic receptors containing α4, α6, β2, and β3 subunits are expressed in midbrain dopaminergic neurons, and they are implicated in the response to smoked nicotine. Here, we have studied the cell biological and biophysical properties of receptors containing α6 and β3 subunits by using fluorescent proteins fused within the M3-M4 intracellular loop. Receptors containing fluorescently tagged β3 subunits were fully functional compared with receptors with untagged β3 subunits. We find that β3- and α6-containing receptors are highly expressed in neurons and that they colocalize with coexpressed, fluorescent α4 and β2 subunits in neuronal soma and dendrites. Förster resonance energy transfer (FRET) reveals efficient, specific assembly of β3 and α6 into nicotinic receptor pentamers of various subunit compositions. Using FRET, we demonstrate directly that only a single β3 subunit is incorporated into nicotinic acetylcholine receptors (nAChRs) containing this subunit, whereas multiple subunit stoichiometries exist for α4- and α6-containing receptors. Finally, we demonstrate that nicotinic ACh receptors are localized in distinct microdomains at or near the plasma membrane using total internal reflection fluorescence (TIRF) microscopy. We suggest that neurons contain large, intracellular pools of assembled, functional nicotinic receptors, which may provide them with the ability to rapidly up-regulate nicotinic responses to endogenous ligands such as ACh, or to exogenous agents such as nicotine. Furthermore, this report is the first to directly measure nAChR subunit stoichiometry using FRET and plasma membrane localization of α6- and β3-containing receptors using TIRF.


Item Type:Article
Related URLs:
URLURL TypeDescription
https://doi.org/10.1124/mol.107.039180DOIArticle
ORCID:
AuthorORCID
Drenan, Ryan M.0000-0002-8141-8577
Lester, Henry A.0000-0002-5470-5255
Additional Information:© 2008 The American Society for Pharmacology and Experimental Therapeutics. Received July 2, 2007; accepted October 11, 2007. We thank members of the Lester laboratory for helpful advice and discussion, including Cagdas Son, Rigo Pantoja, and Fraser Moss. Special thanks to Fraser Moss and Monica Liu for help with molecular biology and Bruce Cohen for help with electrophysiology. This work was supported by the Plum Foundation; National Institutes of Health (NIH) Grants DA017279, DA019375, DA009121, and NS11756; and by Philip Morris International/USA. R.N. was supported by fellowships from the Elizabeth Ross Foundation, University of California Office of the President Tobacco Related Disease Research Program (UCOP TRDRP) Grant 10FT-0174, and the National Alliance for Research on Schizophrenia and Depression. H.J. was supported by the Austrian Science Fund (Fonds zur Förderung der wissenschaftlichen Forschung; Erwin Schrödinger Fellowship J2486). R.M.D. was supported by a fellowship from UCOP TRDRP (15FT-0030) and an NIH National Research Service Award (DA021492).
Funders:
Funding AgencyGrant Number
Plum FoundationUNSPECIFIED
NIHDA017279
NIHDA019375
NIHDA009121
NIHNS11756
Philip Morris InternationalUNSPECIFIED
Elizabeth Ross FoundationUNSPECIFIED
California Tobacco-Related Disease Research Program10FT-0174
National Alliance for Research on Schizophrenia and DepressionUNSPECIFIED
Fonds zur Förderung der wissenschaftlichen Forschung (FWF)J2486
California Tobacco-Related Disease Research Program15FT-0030
NIH Predoctoral FellowshipDA021492
Issue or Number:1
DOI:10.1124/mol.107.039180
Record Number:CaltechAUTHORS:20200422-074016834
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20200422-074016834
Official Citation:Subcellular Trafficking, Pentameric Assembly, and Subunit Stoichiometry of Neuronal Nicotinic Acetylcholine Receptors Containing Fluorescently Labeled α6 and β3 Subunits. Ryan M. Drenan, Raad Nashmi, Princess Imoukhuede, Herwig Just, Sheri McKinney and Henry A. Lester. Molecular Pharmacology January 1, 2008, 73 (1) 27-41; DOI: https://doi.org/10.1124/mol.107.039180
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:102709
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:22 Apr 2020 15:15
Last Modified:16 Nov 2021 18:14

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