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Assembly of α4β2 Nicotinic Acetylcholine Receptors Assessed with Functional Fluorescently Labeled Subunits: Effects of Localization, Trafficking, and Nicotine-Induced Upregulation in Clonal Mammalian Cells and in Cultured Midbrain Neurons

Nashmi, Raad and Dickinson, Mary E. and McKinney, Sheri and Jareb, Mark and Labarca, Cesar and Fraser, Scott E. and Lester, Henry A. (2003) Assembly of α4β2 Nicotinic Acetylcholine Receptors Assessed with Functional Fluorescently Labeled Subunits: Effects of Localization, Trafficking, and Nicotine-Induced Upregulation in Clonal Mammalian Cells and in Cultured Midbrain Neurons. Journal of Neuroscience, 23 (37). pp. 11554-11567. ISSN 0270-6474. PMCID PMC6740951. https://resolver.caltech.edu/CaltechAUTHORS:20160212-092320526

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Abstract

Fura-2 recording of Ca^(2+) influx was used to show that incubation in 1 μM nicotine (2-6 d) upregulates several pharmacological components of acetylcholine (ACh) responses in ventral midbrain cultures, including a MLA-resistant, DHβE-sensitive component that presumably corresponds to α4β2 receptors. To study changes in α4β2 receptor levels and assembly during this upregulation, we incorporated yellow and cyan fluorescent proteins (YFPs and CFPs) into the α4 or β2 M3-M4 intracellular loops, and these subunits were coexpressed in human embryonic kidney (HEK) 293T cells and cultured ventral midbrain neurons. The fluorescent receptors resembled wild-type receptors in maximal responses to ACh, dose-response relations, ACh-induced Ca^(2+) influx, and somatic and dendritic distribution. Transfected midbrain neurons that were exposed to nicotine (1 d) displayed greater levels of fluorescent α4 and β2 nicotinic ACh receptor (nAChR) subunits. As expected from the hetero-multimeric nature of α4β2 receptors, coexpression of the α4-YFP and β2-CFP subunits resulted in robust fluorescence resonance energy transfer (FRET), with a FRET efficiency of 22%. In midbrain neurons, dendritic α4β2 nAChRs displayed greater FRET than receptors inside the soma, and in HEK293T cells, a similar increase was noted for receptors that were translocated to the surface during PKC stimulation. When cultured transfected midbrain neurons were incubated in 1 μMnicotine, there was increased FRET in the cell body, denoting increased assembly of α4β2 receptors. Thus, changes in α4β2 receptor assembly play a role in the regulation of α4β2 levels and responses in both clonal cell lines and midbrain neurons, and the regulation may result from Ca^(2+)-stimulated pathways.


Item Type:Article
Related URLs:
URLURL TypeDescription
https://doi.org/10.1523/jneurosci.23-37-11554.2003DOIArticle
http://www.ncbi.nlm.nih.gov/pmc/articles/pmc6740951/PubMed CentralArticle
ORCID:
AuthorORCID
Fraser, Scott E.0000-0002-5377-0223
Lester, Henry A.0000-0002-5470-5255
Additional Information:© 2003 Society for Neuroscience. For the first six months after publication SfN’s license will be exclusive. Beginning six months after publication the Work will be made freely available to the public on SfN’s website to copy, distribute, or display under a Creative Commons Attribution 4.0 International (CC BY 4.0) license (https://creativecommons.org/licenses/by/4.0/). Received Aug. 13, 2003; revised Oct. 14, 2003; accepted Oct. 16, 2003. This work was supported by National Institutes of Health Grants NS11756 and HD37105 and the California Tobacco-Related Disease Research Program (TRDRP). R.N. was supported by a postdoctoral fellowship from the TRDRP (10FT-0174) and the Elizabeth Ross Fellowship. We thank Bruce Cohen and Julian Revie for critical evaluation of this manuscript, Eric Slimko for providing the GluClβ-YFP GluClα-CFP cDNAs, Donghong Ju, Alexa Mundy, Matthew Abramian, and Kai Sung for technical help, Qi Huang for writing some of the data analysis programs, and James Fisher for performing several three-cube FRET experiments.
Funders:
Funding AgencyGrant Number
NIHNS11756
NIHHD37105
California Tobacco-Related Disease Research Program10FT-0174
Elizabeth Ross FellowshipUNSPECIFIED
Subject Keywords:nicotine addiction; nicotinic receptors; receptor assembly; FRET; α4β2; fluorescent protein
Issue or Number:37
PubMed Central ID:PMC6740951
Record Number:CaltechAUTHORS:20160212-092320526
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20160212-092320526
Official Citation:Assembly of α4β2 Nicotinic Acetylcholine Receptors Assessed with Functional Fluorescently Labeled Subunits: Effects of Localization, Trafficking, and Nicotine-Induced Upregulation in Clonal Mammalian Cells and in Cultured Midbrain Neurons Raad Nashmi, Mary E. Dickinson, Sheri McKinney, Mark Jareb, Cesar Labarca, Scott E. Fraser, and Henry A. Lester The Journal of Neuroscience, 17 December 2003, 23(37):11554-11567
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:64459
Collection:CaltechAUTHORS
Deposited By: Melissa Ray
Deposited On:19 Feb 2016 20:38
Last Modified:24 Apr 2020 18:27

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