Functional expression of nicotinic acetylcholine receptors containing rat alpha 7 subunits in human SH-SY5Y neuroblastoma cells.
Neuronal nicotinic acetylcholine receptors (nAChR) are made from different combinations of subunits encoded by a diverse family of genes. However, the recently cloned alpha 7 gene codes for subunits that can form homooligomeric nAChR complexes when expressed in Xenopus oocytes. Electrophysiological studies reveal that these alpha 7-nAChR function as alpha-bungarotoxin (Bgt)-sensitive, quickly activating/inactivating ion channels with a unique pharmacological profile and an unusually high permeability to calcium ions. Although similar observations have been made in studies of Bgt-sensitive, functional nAChR subtypes that are naturally expressed in neuronal cells, all attempts until now to reconstitute functional alpha 7-nAChR in cell lines have failed. Here we report the successful use of SH-SY5Y human neuroblastoma cells, which naturally express low levels of endogenous alpha 7 transcripts, to stably overexpress heterologous rat nAChR alpha 7 transgenes. These transgenes are expressed as the appropriately-sized alpha 7 messages and protein, and stably transfected SH-SY5Y cells have over 30-times higher levels of specific Bgt binding sites than do wild-type cells. Whole cell current recordings confirm that transfected cells express functional nAChR that are sensitive to blockade by Bgt and display the typical physiological and pharmacological profiles of alpha 7-nAChR. We conclude that stable, functional expression of alpha 7 transgenes in a mammalian cell line has been achieved for the first time.
Division of Neurobiology, Barrow Neurological Institute, Phoenix, AZ 85013.
E
E
Puchacz
B
B
Buisson
D
D
Bertrand
R J
RJ
Lukas
D000818:Animals; D001665:Binding Sites; D002038:Bungarotoxins / Q000378:metabolism / Q000494:pharmacology; D018076:DNA, Complementary / Q000235:genetics; D004594:Electrophysiology; D015870:Gene Expression; D006801:Humans; D009447:Neuroblastoma / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D011978:Receptors, Nicotinic / Q000235:genetics* / Q000502:physiology; D011994:Recombinant Proteins; D014162:Transfection; D014407:Tumor Cells, Cultured
D002038:Bungarotoxins; D018076:DNA, Complementary; D012333:RNA, Messenger; D011978:Receptors, Nicotinic; D011994:Recombinant Proteins