Abstract
Neuronal nicotinic acetylcholine (ACh) receptors are ligand-gated, cation-selective ion channels. Nicotinic receptors containing α4, α6, 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 α6 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 α6-containing receptors are highly expressed in neurons and that they colocalize with coexpressed, fluorescent α4 and β2 subunits in neuronal soma and dendrites. Förster resonance energy transfer (FRET) reveals efficient, specific assembly of β3 and α6 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 α4- and α6-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 α6- and β3-containing receptors using TIRF.
Footnotes
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This work was supported by the Plum Foundation; National Institutes of Health (NIH) Grants DA017279, DA019375, DA009121, and NS11756; and by Philip Morris International/USA. R.N. was supported by fellowships from the Elizabeth Ross Foundation, University of California Office of the President Tobacco Related Disease Research Program (UCOP TRDRP) Grant 10FT-0174, and the National Alliance for Research on Schizophrenia and Depression. H.J. was supported by the Austrian Science Fund (Fonds zur Förderung der wissenschaftlichen Forschung; Erwin Schrödinger Fellowship J2486). R.M.D. was supported by a fellowship from UCOP TRDRP (15FT-0030) and an NIH National Research Service Award (DA021492).
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ABBREVIATIONS: HEK, human embryonic kidney; nAChR, nicotinic acetylcholine receptor; ACh, acetylcholine; YFP, yellow fluorescent protein; CFP, cyan fluorescent protein; FRET, Förster resonance energy transfer; TIRF, total internal reflection fluorescence; PCR, polymerase chain reaction; DMEM, Dulbecco's modified Eagle's medium; WT, wild-type; XFP, yellow or cyan fluorescent protein; NA, numerical aperture; E, FRET efficiency; GluCl, glutamate-gated chloride; HS, high sensitivity; LS, low sensitivity.
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↵ The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.
- Received July 2, 2007.
- Accepted October 11, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
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