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- W2016026256 abstract "The upregulation and preferential assembly of high sensitivity (α4)2(β2)3 neuronal nicotinic acetylcholine receptors (nAChRs) contribute to aspects of nicotine addiction such as sensitization and tolerance. The cellular mechanisms of these events, however, remain elusive. We employed fluorescently tagged nAChRs to study plasma membrane upregulation by total internal reflection fluorescence microscopy (TIRFM) as well as changes in intracellular receptor stoichiometry using pixel-based Forster's resonance energy transfer (FRET). To delineate the effect of β2 on α4 nAChR trafficking in the absence of nicotine, mouse neuroblastoma (N2a) cells were transiently transfected with either α4-meGFP/wildtype β2 or α4-meGFP/wildtype β4 subunits (m = monomeric; e = enhanced) and imaged at 48 h post-transfection by TIRFM. To set TIRFM parameters, cells were co-transfected with the pCS2-mcherry plasmid, which served as a reference probe. pCS2-mcherry expresses mcherry with a lyn kinase membrane localization signal, allowing visualization of the PM using red emission from mcherry. The α4-meGFP reporter was used to detect receptor expression at the PM. Results showed that the α4-meGFP/wildtype β2 receptors trafficked to the PM in ∼10 % of the cells while ∼90 % of imaged cells displayed α4-meGFP/wildtype β4 at the PM. In the presence of nicotine (0.1 μM for 48 h), α4-meGFP/wildtype β2 transfected N2a cells displayed a clear increase in receptor trafficking to the PM when visualized using TIRFM. Pixel-based sensitized emission FRET studies on N2a cells transiently transfected with an α4-mcherry and β2-meGFP FRET pair showed that chronic nicotine exposure (1 μM, 24 h) resulted in an increase in assembly of the high sensitivity (α4)2(β2)3 population of receptors, a phenomenon that was blocked by co-incubation with the competitive nAChR antagonist, DhβE (1 μM). These preliminary results point to a modulatory role of β2 subunits as well as a possibly important role for activity-dependent receptor upregulation." @default.
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- W2016026256 date "2009-02-01" @default.
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- W2016026256 title "Cellular Basis Of Nicotine-induced nAChr Upregulation" @default.
- W2016026256 doi "https://doi.org/10.1016/j.bpj.2008.12.763" @default.
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