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

Abstract

Neuronal nicotinic acetylcholine (ACh) receptors are ligand-gated, cation-selective ion channels. Nicotinic receptors containing alpha4, alpha6, beta2, and beta3 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 alpha6 and beta3 subunits by using fluorescent proteins fused within the M3-M4 intracellular loop. Receptors containing fluorescently tagged beta3 subunits were fully functional compared with receptors with untagged beta3 subunits. We find that beta3- and alpha6-containing receptors are highly expressed in neurons and that they colocalize with coexpressed, fluorescent alpha4 and beta2 subunits in neuronal soma and dendrites. Förster resonance energy transfer (FRET) reveals efficient, specific assembly of beta3 and alpha6 into nicotinic receptor pentamers of various subunit compositions. Using FRET, we demonstrate directly that only a single beta3 subunit is incorporated into nicotinic acetylcholine receptors (nAChRs) containing this subunit, whereas multiple subunit stoichiometries exist for alpha4- and alpha6-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 alpha6- and beta3-containing receptors using TIRF.