 |
The Journal of Neuroscience, December 17, 2003, 23(37):11554-11567
 Previous Article | Next Article 
Cellular/Molecular
Assembly of  4 2 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,1
Mary E. Dickinson,1,2
Sheri McKinney,1
Mark Jareb,1,3
Cesar Labarca,1
Scott E. Fraser,1,2 and
Henry A. Lester1
1Division of Biology, 2Biological Imaging Center, California Institute of Technology, Pasadena, California 91125, and 3Department of Biology, Sacred Heart University, Fairfield, Connecticut 06825
Fura-2 recording of Ca2+ 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, DH E-sensitive component that presumably corresponds to  42 receptors. To study changes in 42 receptor levels and assembly during this upregulation, we incorporated yellow and cyan fluorescent proteins (YFPs and CFPs) into the 4 or 2 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 Ca2+ influx, and somatic and dendritic distribution. Transfected midbrain neurons that were exposed to nicotine (1 d) displayed greater levels of fluorescent 4 and 2 nicotinic ACh receptor (nAChR) subunits. As expected from the hetero-multimeric nature of 42 receptors, coexpression of the 4-YFP and 2-CFP subunits resulted in robust fluorescence resonance energy transfer (FRET), with a FRET efficiency of 22%. In midbrain neurons, dendritic 42 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 µM nicotine, there was increased FRET in the cell body, denoting increased assembly of 42 receptors. Thus, changes in 42 receptor assembly play a role in the regulation of 42 levels and responses in both clonal cell lines and midbrain neurons, and the regulation may result from Ca2+-stimulated pathways.
Key words: nicotine addiction; nicotinic receptors; receptor assembly; FRET; 42; fluorescent protein
Received Aug 13, 2003;
revised October 14, 2003;
accepted October 16, 2003.
This article has been cited by other articles:
|
|
|
|
 
R. M. Drenan, R. Nashmi, P. Imoukhuede, H. Just, S. McKinney, and H. A. Lester
Subcellular Trafficking, Pentameric Assembly, and Subunit Stoichiometry of Neuronal Nicotinic Acetylcholine Receptors Containing Fluorescently Labeled {alpha}6 and 3 Subunits
Mol. Pharmacol.,
January 1, 2008;
73(1):
27 - 41.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
N. Kabbani, M. P. Woll, R. Levenson, J. M. Lindstrom, and J.-P. Changeux
Intracellular complexes of the 2 subunit of the nicotinic acetylcholine receptor in brain identified by proteomics
PNAS,
December 18, 2007;
104(51):
20570 - 20575.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. Nashmi, C. Xiao, P. Deshpande, S. McKinney, S. R. Grady, P. Whiteaker, Q. Huang, T. McClure-Begley, J. M. Lindstrom, C. Labarca, et al.
Chronic Nicotine Cell Specifically Upregulates Functional {alpha}4* Nicotinic Receptors: Basis for Both Tolerance in Midbrain and Enhanced Long-Term Potentiation in Perforant Path
J. Neurosci.,
August 1, 2007;
27(31):
8202 - 8218.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Yamamoto, J. Yamada, S. Ueno, H. Kubota, T. Furukawa, S. Yamamoto, and A. Fukuda
Insertion of {alpha}7 Nicotinic Receptors at Neocortical Layer V GABAergic Synapses Is Induced by a Benzodiazepine, Midazolam
Cereb Cortex,
March 1, 2007;
17(3):
653 - 660.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Kishi and J. H. Steinbach
Role of the Agonist Binding Site in Up-Regulation of Neuronal Nicotinic {alpha}4beta2 Receptors
Mol. Pharmacol.,
December 1, 2006;
70(6):
2037 - 2044.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. Xu, Y. Zhu, and S. F. Heinemann
Identification of sequence motifs that target neuronal nicotinic receptors to dendrites and axons.
J. Neurosci.,
September 20, 2006;
26(38):
9780 - 9793.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. J. Smith, K. Pozo, K. Brickley, and F. A. Stephenson
Mapping the GRIF-1 Binding Domain of the Kinesin, KIF5C, Substantiates a Role for GRIF-1 as an Adaptor Protein in the Anterograde Trafficking of Cargoes
J. Biol. Chem.,
September 15, 2006;
281(37):
27216 - 27228.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. K. Staley, S. Krishnan-Sarin, K. P. Cosgrove, E. Krantzler, E. Frohlich, E. Perry, J. A. Dubin, K. Estok, E. Brenner, R. M. Baldwin, et al.
Human Tobacco Smokers in Early Abstinence Have Higher Levels of beta2* Nicotinic Acetylcholine Receptors than Nonsmokers.
J. Neurosci.,
August 23, 2006;
26(34):
8707 - 8714.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. Fonck, B. N. Cohen, R. Nashmi, P. Whiteaker, D. A. Wagenaar, N. Rodrigues-Pinguet, P. Deshpande, S. McKinney, S. Kwoh, J. Munoz, et al.
Novel Seizure Phenotype and Sleep Disruptions in Knock-In Mice with Hypersensitive {alpha}4* Nicotinic Receptors
J. Neurosci.,
December 7, 2005;
25(49):
11396 - 11411.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. B. Ficklin, S. Zhao, and G. Feng
Ubiquilin-1 Regulates Nicotine-induced Up-regulation of Neuronal Nicotinic Acetylcholine Receptors
J. Biol. Chem.,
October 7, 2005;
280(40):
34088 - 34095.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
B. S. Khakh, J. A. Fisher, R. Nashmi, D. N. Bowser, and H. A. Lester
An Angstrom Scale Interaction between Plasma Membrane ATP-Gated P2X2 and {alpha}4{beta}2 Nicotinic Channels Measured with Fluorescence Resonance Energy Transfer and Total Internal Reflection Fluorescence Microscopy
J. Neurosci.,
July 20, 2005;
25(29):
6911 - 6920.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Y. F. Vallejo, B. Buisson, D. Bertrand, and W. N. Green
Chronic Nicotine Exposure Upregulates Nicotinic Receptors by a Novel Mechanism
J. Neurosci.,
June 8, 2005;
25(23):
5563 - 5572.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. Darsow, T. K. Booker, J. C. Pina-Crespo, and S. F. Heinemann
Exocytic Trafficking Is Required for Nicotine-induced Up-regulation of {alpha}4{beta}2 Nicotinic Acetylcholine Receptors
J. Biol. Chem.,
May 6, 2005;
280(18):
18311 - 18320.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Alkondon and E. X. Albuquerque
Nicotinic Receptor Subtypes in Rat Hippocampal Slices Are Differentially Sensitive to Desensitization and Early in Vivo Functional Up-Regulation by Nicotine and to Block by Bupropion
J. Pharmacol. Exp. Ther.,
May 1, 2005;
313(2):
740 - 750.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
E. Ilegems, H. M. Pick, C. Deluz, S. Kellenberger, and H. Vogel
Noninvasive Imaging of 5-HT3 Receptor Trafficking in Live Cells: FROM BIOSYNTHESIS TO ENDOCYTOSIS
J. Biol. Chem.,
December 17, 2004;
279(51):
53346 - 53352.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. A. Fisher, G. Girdler, and B. S. Khakh
Time-Resolved Measurement of State-Specific P2X2 Ion Channel Cytosolic Gating Motions
J. Neurosci.,
November 17, 2004;
24(46):
10475 - 10487.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. R. Tapper, S. L. McKinney, R. Nashmi, J. Schwarz, P. Deshpande, C. Labarca, P. Whiteaker, M. J. Marks, A. C. Collins, and H. A. Lester
Nicotine Activation of {alpha}4* Receptors: Sufficient for Reward, Tolerance, and Sensitization
Science,
November 5, 2004;
306(5698):
1029 - 1032.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
