An in vitro study of the sub-cellular distribution of nicotinic receptors.
Nicotinic and serotoninergic 5HT3 receptors share important sequence identities except for their cytoplasmic loop. Both ends of this loop display conserved 3D helical structures with distinct primary sequences. We decided to check whether these two helices named F and G play a role in the sub-cellular distribution of different nicotinic receptors. We systematically exchanged each helix with the equivalent sequence of neuronal nicotinic and alpha4, beta2 and alpha7 subunits in the functional chimeric alpha7-5HT3 receptor used as a model system. The new chimeras were expressed in vitro in polarized epithelial cells from pig kidney. We quantified synthesis and export of the receptors to the cell surface by measuring alpha-bungarotoxin binding sites. Immunogold labelling was used, at the electron microscope level, to determine the amount of each chimera present at either domain, apical and/or basolateral, of these cells. We noticed that in epithelial cells the majority of alpha-bungarotoxin binding sites remained sequestered in the cytoplasm as already observed in neurons in vivo. The majority of the pentamers present at the cell surface were located at the apical domain. Our results suggest that helix F and G differently regulate assembly and export to the cell surface of alpha-bungarotoxin binding receptors.
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D001665:Binding Sites; D002038:Bungarotoxins / Q000378:metabolism; D016764:Cell Polarity; D002478:Cells, Cultured; D003593:Cytoplasm / Q000378:metabolism; D004847:Epithelial Cells / Q000648:ultrastructure; D049452:Green Fluorescent Proteins; D006624:Hippocampus / Q000166:cytology / Q000196:embryology; D018374:LLC-PK1 Cells; D008164:Luminescent Proteins / Q000378:metabolism; D009474:Neurons / Q000166:cytology / Q000378:metabolism; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D051381:Rats; D017207:Rats, Sprague-Dawley; D011978:Receptors, Nicotinic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011985:Receptors, Serotonin / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013552:Swine
D000911:Antibodies, Monoclonal; D002038:Bungarotoxins; D008164:Luminescent Proteins; D011978:Receptors, Nicotinic; D011985:Receptors, Serotonin; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins