Functional expression of muscle-type nicotinic acetylcholine receptors in rat forebrain neurons in vitro.
Molecular studies aimed at modifying ligand-gated ion channel function in primary neuronal cultures have been hampered by the limited efficacy of gene transfer techniques. Although viruses can effectively deliver genes to neurons, their preparation requires a substantial effort and their usefulness in delivering multiple subunits of a single protein has not been well established. In this study, we have successfully cotransfected mammalian expression plasmids for all subunits of the muscle nicotinic acetylcholine receptor (nAChR), along with a plasmid containing green fluorescent protein (GFP), into rat forebrain neurons using a calcium phosphate method. Immunostaining experiments and cell counting showed that the nAChR were successfully expressed in approximately 1% of the neurons in the culture. The functionality of the expressed receptors was demonstrated electrophysiologically as carbachol-induced currents were present in nearly 90% of the GFP-positive neurons tested. These currents could be completely inhibited by the irreversible antagonist alpha-cobratoxin. Surprisingly, no currents were obtained in any of the GFP-negative cells tested. These results demonstrate that gene delivery of complex, multi-subunit proteins can be successfully performed in cultured neurons, thereby opening new strategies for the pharmacological study of ligand-gated ion channels.
Santos
S
S
Department of Neurobiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.
Aizenman
E
E
D000818:Animals; D017930:Genes, Reporter / Q000235:genetics; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics; D009474:Neurons / Q000378:metabolism*; D016548:Prosencephalon / Q000166:cytology / Q000196:embryology / Q000378:metabolism*; D051381:Rats; D011978:Receptors, Nicotinic / Q000096:biosynthesis* / Q000235:genetics; D014162:Transfection
D008164:Luminescent Proteins; D011978:Receptors, Nicotinic; D049452:Green Fluorescent Proteins