Host cell-specific folding of the neuronal nicotinic receptor alpha8 subunit.
Heterologous expression of the neuronal nicotinic acetylcholine receptor alpha8 subunit in cultured mammalian cell lines has revealed that the correct folding of this protein is dependent on the host cell type. The alpha8 subunit, which is able to form homo-oligomeric ion channels when expressed in Xenopus oocytes, could be detected in all transfected cell lines by both immunoprecipitation and immunofluorescence microscopy with a monoclonal antibody that recognises a linear epitope. In contrast, the alpha8 subunit could be detected in some but not in all transfected cell lines with a monoclonal antibody that recognises a conformation-sensitive epitope or by nicotinic radioligand binding. It is interesting that although correctly folded alpha8 protein could be detected in transfected rat pituitary (GH4C1) cells, only misfolded alpha8 protein could be detected in a large subpopulation of transfectants (transient or clonal stable isolates). We have also found that the protein encoded by a chimaeric cDNA (constructed from the N-terminal region of alpha8 and the C-terminal domain of the serotonin 5-HT3 receptor subunit) is expressed efficiently, and in a conformation that binds alpha-bungarotoxin, in all cell types examined. These results, together with previous expression studies with the homo-oligomeric alpha7 subunit and hetero-oligomeric nicotinic receptor subunit combinations, suggest that the cell-specific folding described here is a phenomenon that may be characteristic of homo-oligomeric nicotinic receptors.
Cooper
S T
ST
Department of Pharmacology, University College London, England, UK.
Millar
N S
NS
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D002460:Cell Line; D002499:Centrifugation, Density Gradient; D002645:Chickens; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000196:embryology / Q000378:metabolism; D046911:Macromolecular Substances; D008856:Microscopy, Fluorescence; D009447:Neuroblastoma / Q000378:metabolism / Q000473:pathology; D009474:Neurons / Q000378:metabolism; D010902:Pituitary Gland / Q000166:cytology / Q000378:metabolism; D011233:Precipitin Tests; D011487:Protein Conformation; D017510:Protein Folding; D011869:Radioligand Assay; D051381:Rats; D011978:Receptors, Nicotinic / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D011985:Receptors, Serotonin / Q000235:genetics; D044406:Receptors, Serotonin, 5-HT3; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D013045:Species Specificity; D013395:Sucrose / Q000737:chemistry; D014162:Transfection; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D046911:Macromolecular Substances; D011978:Receptors, Nicotinic; D011985:Receptors, Serotonin; D044406:Receptors, Serotonin, 5-HT3; D011993:Recombinant Fusion Proteins; D013395:Sucrose