title: Nicotine up-regulates alpha4beta2 nicotinic receptors and ER exit sites via stoichiometry-dependent chaperoning.
abstract: The up-regulation of α4β2* 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 α4 nAChR subunits and an FP-tagged Sec24D endoplasmic reticulum (ER) exit site marker. Total internal reflection fluorescence microscopy shows that nicotine (0.1 µM for 48 h) up-regulates α4β2 nAChRs at the plasma membrane (PM), despite increasing the fraction of α4β2 nAChRs that remain in near-PM ER. Pixel-resolved normalized Förster resonance energy transfer microscopy between α4-FP subunits shows that nicotine stabilizes the (α4)(2)(β2)(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 β2(enhanced-ER-export) mutant subunit that mimics two regions of the β4 subunit sequence: the presence of an ER export motif and the absence of an ER retention/retrieval motif. The α4β2(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 α4β2(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 α4β2 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 β2(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.
authors.affiliation: Division of Biology MC 156-29, California Institute of Technology, Pasadena, CA 91125, USA.
authors.forename: Rahul
authors.initials: R
authors.lastname: Srinivasan
authors.forename: Rigo
authors.initials: R
authors.lastname: Pantoja
authors.forename: Fraser J
authors.initials: FJ
authors.lastname: Moss
authors.forename: Elisha D W
authors.initials: ED
authors.lastname: Mackey
authors.forename: Cagdas D
authors.initials: CD
authors.lastname: Son
authors.forename: Julie
authors.initials: J
authors.lastname: Miwa
authors.forename: Henry A
authors.initials: HA
authors.lastname: Lester
mesh_terms: D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D049452:Green Fluorescent Proteins / Q000378:metabolism; D051379:Mice; D008853:Microscopy / Q000379:methods; D008856:Microscopy, Fluorescence / Q000379:methods; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism*; D009538:Nicotine / Q000494:pharmacology*; D021122:Protein Subunits; D021381:Protein Transport / Q000187:drug effects; D011978:Receptors, Nicotinic / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D014029:Tobacco Use Disorder / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D015854:Up-Regulation / Q000187:drug effects; D033921:Vesicular Transport Proteins / Q000235:genetics / Q000378:metabolism; D021601:trans-Golgi Network / Q000378:metabolism
chemical_list: D018832:Molecular Chaperones; D021122:Protein Subunits; D011978:Receptors, Nicotinic; C530641:SEC24D protein, human; D033921:Vesicular Transport Proteins; C403531:nicotinic receptor alpha4beta2; D049452:Green Fluorescent Proteins; D009538:Nicotine