Abstract
We investigated assembly and function of nicotinic acetylcholine receptors (nAChRs) composed of α7 and β2 subunits. We measured optical and electrophysiological properties of wild-type and mutant subunits expressed in cell lines and Xenopus laevis oocytes. Laser scanning confocal microscopy indicated that fluorescently tagged α7 and β2 subunits colocalize. Förster resonance energy transfer between fluorescently tagged subunits strongly suggested that α7 and β2 subunits coassemble. Total internal reflection fluorescence microscopy revealed that assemblies localized to filopodia-like processes of SH-EP1 cells. Gain-of-function α7 and β2 subunits confirmed that these subunits coassemble within functional receptors. Moreover, α7β2 nAChRs composed of wild-type subunits or fluorescently tagged subunits had pharmacological properties similar to those of α7 nAChRs, although amplitudes of α7β2 nAChR-mediated, agonist-evoked currents were generally ∼2-fold lower than those for α7 nAChRs. It is noteworthy that α7β2 nAChRs displayed sensitivity to low concentrations of the antagonist dihydro-β-erythroidine that was not observed for α7 nAChRs at comparable concentrations. In addition, cysteine mutants revealed that the α7-β2 subunit interface does not bind ligand in a functionally productive manner, partly explaining lower α7β2 nAChR current amplitudes and challenges in identifying the function of native α7β2 nAChRs. On the basis of our findings, we have constructed a model predicting receptor function that is based on stoichiometry and position of β2 subunits within the α7β2 nAChRs.
Footnotes
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The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.This work was supported by the National Institutes of Health National Institute on Drug Abuse [Grants DA015389, DA027070, DA012242]; the National Institutes of Health National Institute of Neurological Diseases and Stroke [Grant NS11756]; the National Institutes of Health National Institute of Mental Health [Grant MH086383]; the National Institutes of Health National Institute of General Medical Sciences [Grant GM057481]; a US National Science Foundation Graduate Research Fellowship; the Barrow Neurological Foundation; a Catholic Healthcare West SEED Grant; the Biodesign Institute at Arizona State University, the Swiss National Science Foundation; the EC Neurocypres Grant; and the California Tobacco-Related Disease Research Program.
Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
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ABBREVIATIONS:
- nAChR
- nicotinic acetylcholine receptor
- MS/DB
- medial septum-diagonal band
- YFP
- yellow fluorescent protein
- CFP
- cyan fluorescent protein
- GluCl
- glutamate-gated chloride channel
- α4Y
- YFP-tagged nAChR α4 subunit; α7C YFP-tagged nAChR α7 subunit
- α7Y
- CFP-tagged nAChR α7 subunit
- β2C
- CFP-tagged nAChR β2 subunit
- β2Ch
- mCherry-tagged nAChR β2 subunit
- GCαY
- YFP-tagged glutamate-gated chloride channel α subunit
- GCβC
- CFP-tagged glutamate-gated chloride channel β subunit
- LSCM
- laser scanning confocal microscopy
- FRET
- Förster resonance energy transfer
- TIRF
- total internal reflection fluorescence
- BAPTA
- 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid
- MTSEA
- methanethiosulfonate ethylammonium
- ACh
- acetylcholine
- MLA
- methyllycaconitine
- FP
- fusion protein
- ER
- endoplasmic reticulum
- ROI
- region of interest
- DHβE
- dihydro-β-erythroidine
- PNU-282987
- N-(3R)-1-azabicyclo[2.2.2]oct-3-yl-4-chlorobenzamide
- E
- FRET efficiency
- ID
- intensity of donor FP after photodestruction of acceptor
- IDA
- intensity of donor FP in the presence of the unbleached acceptor
- In
- normalized fluorescence intensity.
- Received June 12, 2011.
- Accepted October 28, 2011.
- Copyright © 2012 The American Society for Pharmacology and Experimental Therapeutics
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