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The Journal of Neuroscience, December 17, 2003, 23(37):11554-11567

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Cellular/Molecular
Assembly of {alpha}4{beta}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{beta}E-sensitive component that presumably corresponds to {alpha}4{beta}2 receptors. To study changes in {alpha}4{beta}2 receptor levels and assembly during this upregulation, we incorporated yellow and cyan fluorescent proteins (YFPs and CFPs) into the {alpha}4 or {beta}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 {alpha}4 and {beta}2 nicotinic ACh receptor (nAChR) subunits. As expected from the hetero-multimeric nature of {alpha}4{beta}2 receptors, coexpression of the {alpha}4-YFP and {beta}2-CFP subunits resulted in robust fluorescence resonance energy transfer (FRET), with a FRET efficiency of 22%. In midbrain neurons, dendritic {alpha}4{beta}2 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 {alpha}4{beta}2 receptors. Thus, changes in {alpha}4{beta}2 receptor assembly play a role in the regulation of {alpha}4{beta}2 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; {alpha}4{beta}2; fluorescent protein

Received Aug 13, 2003; revised October 14, 2003; accepted October 16, 2003.




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