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

• Published in: The Journal of neuroscience Vol. 23; no. 37; pp. 11554 - 11567
• Main Authors: Nashmi, R, Dickinson, ME, McKinney, S, Jareb, M, Labarca, C, Fraser, SE, Lester, HA
• Format: Journal Article
• Language: English
• Published: 17.12.2003
• ISSN: 0270-6474

Fura-2 recording of Ca super(2+) influx was used to show that incubation in 1 mu M nicotine (2-6 d) upregulates several pharmacological components of acetylcholine (ACh) responses in ventral midbrain cultures, including a MLA-resistant, DHssE-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 Ca super(2+) 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 mu M 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 Ca super(2+)-stimulated pathways.