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The 5-HT_3AB Receptor Shows an A_3B_2 Stoichiometry at the Plasma Membrane

Miles, Timothy F. and Dougherty, Dennis A. and Lester, Henry A. (2013) The 5-HT_3AB Receptor Shows an A_3B_2 Stoichiometry at the Plasma Membrane. Biophysical Journal, 105 (4). pp. 887-898. ISSN 0006-3495. PMCID PMC3752109. doi:10.1016/j.bpj.2013.07.015.

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The 5-HT_3AB receptor is the best-characterized heteropentameric 5-HT_3 receptor. Under conditions of heterologous expression, the 5-HT_3AB receptor shows a single functionally resolvable population, suggesting the presence of a unique subunit stoichiometry; however, conflicting previous reports have suggested two different possible stoichiometries. Here we isolate plasma membrane sheets containing assembled receptors from individual HEK293T cells. We then determine the stoichiometry of 5-HT_3AB receptors on the plasma membrane by fluorescence methods, employing meCFP- and meYFP-labeled A and B subunits. Over a wide range of cDNA transfection ratios, fluorescence intensity ratios are closest to values that correspond to a subunit ratio of A_3B_2. Förster resonance energy transfer (family FRET) efficiencies provide minor corrections (3–6%) to the subunit ratios and provide independent support for a predominantly A_3B_2 stoichiometry on the plasma membrane sheets. Twin FRET efficiencies support these data, also suggesting that the two B subunits are nonadjacent in most of the heteropentamers. The high-frequency variant HTR3B p.Y129S (c.386A>C, rs11767445), linked to psychiatric disease, also forms A_3B_2 receptors on the plasma membrane. The 5-HT_3B Y129S, subunit incorporates in a slightly (11–14%) more efficient manner than the common variant. In general, most of the subunits reside within the cell. In contrast to the findings for the plasma membrane, the relative abundances and FRET characteristics of intracellular subunits depend strongly on the transfection ratio. The straightforward and unambiguous combination of plasma membrane-sheet isolation, fluorescence intensity ratios, and FRET is a generally promising procedure for determining stoichiometry of proteins on the plasma membrane.

Item Type:Article
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URLURL TypeDescription CentralArticle
Dougherty, Dennis A.0000-0003-1464-2461
Lester, Henry A.0000-0002-5470-5255
Additional Information:© 2013 Biophysical Society. Submitted May 16, 2013, and accepted for publication July 11, 2013. Editor: Cynthia Czajkowski. The authors thank Rahul Srinivasan, Kristina N. Daeffler, and Ethan B. Van Arnam for technical assistance and helpful discussion. This work was supported by grants (No. NS034407, No. DA017279, and No. AG033954) from the National Institutes of Health, Bethesda, MD.
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Issue or Number:4
PubMed Central ID:PMC3752109
Record Number:CaltechAUTHORS:20130912-145808068
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Official Citation:Timothy F. Miles, Dennis A. Dougherty, Henry A. Lester, The 5-HT3AB Receptor Shows an A3B2 Stoichiometry at the Plasma Membrane, Biophysical Journal, Volume 105, Issue 4, 20 August 2013, Pages 887-898, ISSN 0006-3495, (
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:41299
Deposited By: Ruth Sustaita
Deposited On:12 Sep 2013 22:29
Last Modified:10 Nov 2021 04:28

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