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Subcellular Trafficking, Pentameric Assembly, and Subunit Stoichiometry of Neuronal Nicotinic Acetylcholine Receptors Containing Fluorescently Labeled 6 and β3 Subunits

Division of Biology, California Institute of Technology, Pasadena, California

Neuronal nicotinic acetylcholine (ACh) receptors are ligand-gated, cation-selective ion channels. Nicotinic receptors containing 4, 6, 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 6 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 6-containing receptors are highly expressed in neurons and that they colocalize with coexpressed, fluorescent 4 and β2 subunits in neuronal soma and dendrites. Förster resonance energy transfer (FRET) reveals efficient, specific assembly of β3 and 6 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 4- and 6-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 6- and β3-containing receptors using TIRF.

Address correspondence to: Dr. Henry A. Lester, California Institute of Technology, Division of Biology, M/C 156-29, 1200 E. California Blvd., Pasadena, CA 91125. E-mail: lester{at}caltech.edu

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