Localization of Plasma Membrane and Intracellular Neuronal Nicotinic Acetylcholine Receptors Using Quantitative...

Quantification of neuronal nicotinic acetylcholine receptors (nAChRs) expressed on the plasma membrane of mammalian cells is important for the development of novel target-selective therapeutics and for studying chaperone proteins that modulate receptor trafficking. This study presents a robust set of methodologies to express both homomeric and heteromeric nAChRs in mammalian Neuro2a (N2a) cells and to quantify their surface versus intracellular localization using pH-sensitive fluorescent tags. pHuji and superecliptic pHluorin (SEP) are pH-sensitive proteins that can be engineered to tag recombinant proteins, allowing visualization of the spatial distribution of nAChR subunits. These fluorescent tags emit at neutral pH but are quenched in acidic environments. To track the relative internal versus plasma membrane localization of α7, α4, and β2 nAChR subunits, either pHuji or SEP is attached to the C-terminus of each DNA construct. To express α7-pHuji together with its chaperone NACHO, or (α4-SEP; β2-pHuji) in N2a cells, a lipid-based DNA transfection reagent is used. The transfection reagent and plasmid DNA are incubated separately in reduced-serum medium at room temperature, then combined to form lipid-DNA complexes that facilitate plasmid delivery into cells. Following incubation, cells are imaged using live-cell confocal microscopy under both high- and low-pH conditions or in the presence of a subtype-selective fluorescent ligand. Images are analyzed using corrected total cell fluorescence in the relevant fluorophore channels, providing quantitative information on the distribution of fluorescently tagged nAChR subunits between the plasma membrane and intracellular compartments. Live-cell confocal imaging enables real-time tracking of nAChR subunit localization to distinguish differences between internal and surface receptor populations. Robust plasma membrane expression of α7-pHuji or (α4-SEP; β2-pHuji) is achieved within 24 h at 37 °C. This method can be adapted to include additional chaperones, auxiliary subunits, or fluorescent ligands for the study of nAChR trafficking, pharmacological modulation, and disease-related alterations in nAChR expression.