Subcellular trafficking, pentameric assembly, and subunit stoichiometry of neuronal nicotinic acetylcholine receptors containing fluorescently labeled α6 and β3 Subunits

Neuronal nicotinic acetylcholine (ACh) receptors are ligand-gated, cation-selective ion channels. Nicotinic receptors containing alpha 4, alpha 6, beta 2, and beta 3 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 alpha 6 and beta 3 subunits by using fluorescent proteins fused within the M3-M4 intracellular loop. Receptors containing fluorescently tagged beta 3 subunits were fully functional compared with receptors with untagged beta 3 subunits. We find that beta 3- and alpha 6- containing receptors are highly expressed in neurons and that they colocalize with coexpressed, fluorescent alpha 4 and beta 2 subunits in neuronal soma and dendrites. Forster resonance energy transfer (FRET) reveals efficient, specific assembly of beta 3 and alpha 6 into nicotinic receptor pentamers of various subunit compositions. Using FRET, we demonstrate directly that only a single beta 3 subunit is incorporated into nicotinic acetylcholine receptors (nAChRs) containing this subunit, whereas multiple subunit stoichiometries exist for alpha 4- and alpha 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 alpha 6- and beta 3- containing receptors using TIRF.