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

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