Received for publication July 5, 2007.
Revised October 8, 2007.
Accepted for publication October 11, 2007.

Subcellular Trafficking, Pentameric Assembly and Subunit Stoichiometry of Neuronal Nicotinic ACh Receptors Containing Fluorescently-Labeled 6 and 3 Subunits

Abstract

Neuronal nicotinic ACh receptors are ligand-gated, cation-selective ion channels. Nicotinic receptors containing 4, 6, 2, and 3 subunits are expressed in midbrain dopaminergic neurons and are implicated in the response to smoked nicotine. Here we have studied the cell biological and biophysical properties of receptors containing 6 and 3 subunits by utilizing fluorescent proteins fused within the M3-M4 intracellular loop. Receptors containing fluorescently-tagged 3 subunits were fully functional compared to receptors with untagged 3 subunits. We find that 3 and 6-containing receptors are highly expressed in neurons, and co-localize with co-expressed, fluorescent 4 and 2 subunits in neuronal soma and dendrites. Forster resonance energy transfer (FRET) reveals efficient, specific assembly of 3 and 6 into nicotinic receptor pentamers of various subunit compositions. Using FRET, we demonstrate directly that only a single 3 subunit is incorporated into 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. Further, this report is the first to directly measure nAChR subunit stoichiometry using FRET and plasma membrane localization of 6 and 3-containing receptors using TIRF

Key words: Nicotinic cholinergic, Func. analysis receptor/ion channel mutants, Fluorescence techniques, Mutagenesis/Chimeric approaches, Drug tolerance/dependence

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