Nicotine up-regulates alpha 4 beta 2 nicotinic receptors and ER exit sites via stoichiometry-dependent chaperoning

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Date

2011-01-01

Author

Srinivasan, Rahul
Pantoja, Rigo
Moss, Fraser J.
Mackey, Elisha D. W.
Son, Çağdaş Devrim
Miwa, Julie
Lester, Henry A.

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The up-regulation of alpha 4 beta 2* nicotinic acetylcholine receptors (nAChRs) by chronic nicotine is a cell-delimited process and may be necessary and sufficient for the initial events of nicotine dependence. Clinical literature documents an inverse relationship between a person's history of tobacco use and his or her susceptibility to Parkinson's disease; this may also result from up-regulation. This study visualizes and quantifies the subcellular mechanisms involved in nicotine-induced nAChR up-regulation by using transfected fluorescent protein (FP)-tagged alpha 4 nAChR subunits and an FP-tagged Sec24D endoplasmic reticulum (ER) exit site marker. Total internal reflection fluorescence microscopy shows that nicotine (0.1 mu M for 48 h) up-regulates alpha 4 beta 2 nAChRs at the plasma membrane (PM), despite increasing the fraction of alpha 4 beta 2 nAChRs that remain in near-PM ER. Pixel-resolved normalized Forster resonance energy transfer microscopy between alpha 4-FP subunits shows that nicotine stabilizes the (alpha 4)(2)(beta 2)(3) stoichiometry before the nAChRs reach the trans-Golgi apparatus. Nicotine also induces the formation of additional ER exit sites (ERES). To aid in the mechanistic analysis of these phenomena, we generated a beta 2(enhanced-ER-export) mutant subunit that mimics two regions of the alpha 4 subunit sequence: the presence of an ER export motif and the absence of an ER retention/retrieval motif. The alpha 4 beta 2(enhanced-ER-export) nAChR resembles nicotine-exposed nAChRs with regard to stoichiometry, intracellular mobility, ERES enhancement, and PM localization. Nicotine produces only small additional PM up-regulation of alpha 4 beta 2(enhanced-ER-export receptors). The experimental data are simulated with a model incorporating two mechanisms: (1) nicotine acts as a stabilizing pharmacological chaperone for nascent alpha 4 beta 2 nAChRs in the ER, eventually increasing PM receptors despite a bottleneck(s) in ER export; and (2) removal of the bottleneck (e. g., by expression of the beta 2(enhanced-ER-export) subunit) is sufficient to increase PM nAChR numbers, even without nicotine. The data also suggest that pharmacological chaperoning of nAChRs by nicotine can alter the physiology of ER processes.

Subject Keywords

Physiology

URI

https://hdl.handle.net/11511/47566

Journal

JOURNAL OF GENERAL PHYSIOLOGY

DOI

https://doi.org/10.1085/jgp.201010532

Collections

Department of Biology, Article

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Citation Formats

R. Srinivasan et al., “Nicotine up-regulates alpha 4 beta 2 nicotinic receptors and ER exit sites via stoichiometry-dependent chaperoning,” JOURNAL OF GENERAL PHYSIOLOGY, pp. 59–79, 2011, Accessed: 00, 2020. [Online]. Available: https://hdl.handle.net/11511/47566.