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Trafficking of α4^* Nicotinic Receptors Revealed by Superecliptic Phluorin

Richards, Christopher I. and Srinivasan, Rahul and Xiao, Cheng and Mackey, Elisha D. W. and Miwa, Julie M. and Lester, Henry A. (2011) Trafficking of α4^* Nicotinic Receptors Revealed by Superecliptic Phluorin. Journal of Biological Chemistry, 286 (36). pp. 31241-31249. ISSN 0021-9258. PMCID PMC3173132. doi:10.1074/jbc.M111.256024.

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We employed a pH-sensitive GFP analog, superecliptic phluorin, to observe aspects of nicotinic acetylcholine receptor (nAChR) trafficking to the plasma membrane (PM) in cultured mouse cortical neurons. The experiments exploit differences in the pH among endoplasmic reticulum (ER), trafficking vesicles, and the extracellular solution. The data confirm that few α4β4 nAChRs, but many α4β2 nAChRs, remain in neutral intracellular compartments, mostly the ER. We observed fusion events between nAChR-containing vesicles and PM; these could be quantified in the dendritic processes. We also studied the β4R348C polymorphism, linked to amyotrophic lateral sclerosis (ALS). This mutation depressed fusion rates of α4β4 receptor-containing vesicles with the PM by ∼2-fold, with only a small decrease in the number of nAChRs per vesicle. The mutation also decreased the number of ER exit sites, showing that the reduced receptor insertion results from a change at an early stage in trafficking. We confirm the previous report that the mutation leads to reduced agonist-induced currents; in the cortical neurons studied, the reduction amounts to 2–3-fold. Therefore, the reduced agonist-induced currents are caused by the reduced number of α4β4-containing vesicles reaching the membrane. Chronic nicotine exposure (0.2 μm) did not alter the PM insertion frequency or trafficking behavior of α4β4-laden vesicles. In contrast, chronic nicotine substantially increased the number of α4β2-containing vesicle fusions at the PM; this stage in α4β2 nAChR up-regulation is presumably downstream from increased ER exit. Superecliptic phluorin provides a tool to monitor trafficking dynamics of nAChRs in disease and addiction.

Item Type:Article
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URLURL TypeDescription CentralArticle
Xiao, Cheng0000-0001-9649-7450
Lester, Henry A.0000-0002-5470-5255
Additional Information:© 2011 by The American Society for Biochemistry and Molecular Biology, Inc. Received for publication, April 29, 2011, and in revised form, July 13, 2011. This work was supported, in whole or in part, by National Institutes of Health Grants AG033954, DA17279, and NS11756. This work was also supported by the California Tobacco-related Disease Research Program Grant 19KT-0032 and by a gift from Louis and Janet Fletcher. Supported by National Institutes of Health Kirschstein NRSA DA030877 and a Beckman Institute fellowship. Supported by a Tobacco-related Disease Research Program Postdoctoral Fellowship 18FT-0066 and by a Rapid Response grant from the Michael J. Fox Foundation.
Funding AgencyGrant Number
California Tobacco-Related Disease Research Program19KT-0032
Louis and Janet FletcherUNSPECIFIED
NIH Predoctoral FellowshipDA030877
Caltech Beckman InstituteUNSPECIFIED
California Tobacco-Related Disease Research Program18FT-0066
Michael J. Fox FoundationUNSPECIFIED
Arnold and Mabel Beckman FoundationUNSPECIFIED
Issue or Number:36
PubMed Central ID:PMC3173132
Record Number:CaltechAUTHORS:20110922-112958690
Persistent URL:
Official Citation:Trafficking of α4* Nicotinic Receptors Revealed by Superecliptic Phluorin: EFFECTS OF A β4 AMYOTROPHIC LATERAL SCLEROSIS-ASSOCIATED MUTATION AND CHRONIC EXPOSURE TO NICOTINE Christopher I. Richards, Rahul Srinivasan, Cheng Xiao, Elisha D. W. Mackey, Julie M. Miwa, and Henry A. Lester J. Biol. Chem. 2011 286: 31241-31249. First Published on July 18, 2011, doi:10.1074/jbc.M111.256024
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:25403
Deposited By: Ruth Sustaita
Deposited On:22 Sep 2011 19:56
Last Modified:09 Nov 2021 16:32

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