Journal article

Noninvasive imaging of 5-HT3 receptor trafficking in live cells: From biosynthesis to endocytosis

Sequential stages in the life cycle of the ionotropic 5-HT3 receptor (5-HT3R) were resolved temporally and spatially in live cells by multicolor fluorescence confocal microscopy. The insertion of the enhanced cyan fluorescent protein into the large intracellular loop delivered a fluorescent 5-HT3R fully functional in terms of ligand binding specificity and channel activity, which allowed for the first time a complete real-time visualization and documentation of intracellular biogenesis, membrane targeting, and ligand-mediated internalization of a receptor belonging to the ligand-gated ion channel superfamily. Fluorescence signals of newly expressed receptors were detectable in the endoplasmic reticulum about 3 h after transfection onset. At this stage receptor subunits assembled to form active ligand binding sites as demonstrated in situ by binding of a fluorescent 5-HT3R-specific antagonist. After novel protein synthesis was chem. blocked, the 5-HT3 R populations in the endoplasmic reticulum and Golgi cisternae moved virtually quant. to the cell surface, indicating efficient receptor folding and assembly. Intracellular 5-HT3 receptors were trafficking in vesicle-like structures along microtubules to the cell surface at a velocity generally below 1 mm/s and were inserted into the plasma membrane in a characteristic cluster distribution overlapping with actin-rich domains. Internalization of cell surface 5-HT3 receptors was obsd. within minutes after exposure to an extracellular agonist. The authors' orchestrated use of spectrally distinguishable fluorescent labels for the receptor, its cognate ligand, and specific organelle markers can be regarded as a general approach allowing subcellular insights into dynamic processes of membrane receptor trafficking. [on SciFinder (R)]


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