title: The 5-HT3AB receptor shows an A3B2 stoichiometry at the plasma membrane.
abstract: The 5-HT3AB receptor is the best-characterized heteropentameric 5-HT3 receptor. Under conditions of heterologous expression, the 5-HT3AB 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-HT3AB 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 A3B2. Förster resonance energy transfer (family FRET) efficiencies provide minor corrections (3-6%) to the subunit ratios and provide independent support for a predominantly A3B2 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 A3B2 receptors on the plasma membrane. The 5-HT3B 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.
authors: Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, USA.
authors: Timothy F
authors: TF
authors: Miles
authors: Dennis A
authors: DA
authors: Dougherty
authors: Henry A
authors: HA
authors: Lester
mesh_terms: D002462:Cell Membrane / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D057809:HEK293 Cells; D006801:Humans; D021122:Protein Subunits / Q000737:chemistry* / Q000378:metabolism*; D044406:Receptors, Serotonin, 5-HT3 / Q000737:chemistry / Q000378:metabolism*
chemical_list: D021122:Protein Subunits; D044406:Receptors, Serotonin, 5-HT3
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