Nicotinic acetylcholine receptors (nAChRs) assemble in the endoplasmic reticulum (ER) and traffic to the cell surface as pentamers composed of α and β subunits. Many nAChR subtypes can assemble with varying subunit ratios, giving rise to multiple stoichiometries exhibiting different subcellular localization and functional properties. In addition to the endogenous neurotransmitter acetylcholine, nicotine also binds and activates nAChRs and influences their trafficking and expression on the cell surface. Currently, no available technique can specifically elucidate the stoichiometry of nAChRs in the ER versus those in the plasma membrane. Here, we report a method involving single-molecule fluorescence measurements to determine the structural properties of these membrane proteins after isolation in nanoscale vesicles derived from specific organelles. These cell-derived nanovesicles allowed us to separate single membrane receptors while maintaining them in their physiological environment. Sorting the vesicles according to the organelle of origin enabled us to determine localized differences in receptor structural properties, structural influence on transport between organelles, and changes in receptor assembly within intracellular organelles. These organelle-specific nanovesicles revealed that one structural isoform of the α4β2nAChR was preferentially trafficked to the cell surface. Moreover, nicotine altered nAChR assembly in the ER, resulting in increased production of the receptor isoform that traffics more efficiently to the cell surface.Weconclude that the combined effects of the increased assembly of one nAChR stoichiometry and its preferential trafficking likely drive the up-regulation of nAChRs on the cell surface upon nicotine exposure.
|Number of pages
|Journal of Biological Chemistry
|Published - Dec 22 2017
Bibliographical noteFunding Information:
This work was supported in part by the National Institute on Drug Abuse, National Institutes of Health Training Grants DA016176 (A. M. F.-L.) and DA038817. The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology