Inducible, heterologous expression of human alpha7-nicotinic acetylcholine receptors in a native nicotinic receptor-null human clonal line.
Tetracycline-regulated expression of recombinant nicotinic acetylcholine receptors (nAChR) composed of human alpha7 subunits is achieved in native nAChR-null SH-EP1 human epithelial cells. alpha7 subunits are heterologously expressed as messenger RNA and as components of 125I-labeled alpha-bungarotoxin (I-Bgt)-binding nAChR ( approximately 10 pmol per milligram of membrane protein) at levels sensitive to the amount of tetracycline in cell growth medium. I-Bgt-binding alpha7-nAChR appear on the cell surface pool and in intracellular pools. The pharmacological profile for drug competition toward I-Bgt binding to these recombinant alpha7-nAChR matches that of human native alpha7-nAChR naturally expressed in SH-SY5Y human neuroblastoma cells (rank order potency methyllycaconitine>1, 1-dimethyl-4-phenylpiperazinium>(-)nicotine>cytisine>carbamylch oli ne> /=d-tubocurarine). Chronic exposure to nicotine induces up-regulation of human recombinant alpha7-nAChR (80% up-regulation at 10 microM nicotine) just as it does native alpha7-nAChR in other human cell lines. These studies confirm expression of nAChR as homooligomers of human alpha7 subunits from transgenes, establish a native nAChR-null background for such expression, and demonstrate that this expression can be regulated to facilitate studies of human alpha7-nAChR.
Peng
J H
JH
Division of Neurobiology, Barrow Neurological Institute, 350 West Thomas Road, Phoenix, AZ 85013, USA.
Lucero
L
L
Fryer
J
J
Herl
J
J
Leonard
S S
SS
Lukas
R J
RJ
D000109:Acetylcholine / Q000502:physiology; D000157:Aconitine / Q000031:analogs & derivatives / Q000494:pharmacology; D000470:Alkaloids / Q000494:pharmacology; D001392:Azocines; D015152:Blotting, Northern; D002038:Bungarotoxins / Q000494:pharmacology; D002217:Carbachol / Q000494:pharmacology; D018076:DNA, Complementary; D004246:Dimethylphenylpiperazinium Iodide / Q000494:pharmacology; D015870:Gene Expression / Q000187:drug effects / Q000502:physiology; D006801:Humans; D007306:Insecticides / Q000494:pharmacology; D007457:Iodine Radioisotopes; D009447:Neuroblastoma; D009474:Neurons / Q000737:chemistry* / Q000502:physiology*; D009538:Nicotine / Q000494:pharmacology; D018722:Nicotinic Agonists / Q000494:pharmacology; D010957:Plasmids; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011807:Quinolizines; D012333:RNA, Messenger / Q000032:analysis; D011978:Receptors, Nicotinic / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013752:Tetracycline / Q000494:pharmacology; D014162:Transfection; D014403:Tubocurarine / Q000494:pharmacology; D014407:Tumor Cells, Cultured / Q000737:chemistry / Q000166:cytology; D015854:Up-Regulation / Q000235:genetics; D064569:alpha7 Nicotinic Acetylcholine Receptor; D000093843:Quinolizidine Alkaloids
D000109:Acetylcholine; D000157:Aconitine; D000470:Alkaloids; D001392:Azocines; D002038:Bungarotoxins; D002217:Carbachol; D018076:DNA, Complementary; D004246:Dimethylphenylpiperazinium Iodide; D007306:Insecticides; D007457:Iodine Radioisotopes; D009538:Nicotine; D018722:Nicotinic Agonists; D011500:Protein Synthesis Inhibitors; D011807:Quinolizines; D012333:RNA, Messenger; D011978:Receptors, Nicotinic; D011994:Recombinant Proteins; D013752:Tetracycline; D014403:Tubocurarine; D064569:alpha7 Nicotinic Acetylcholine Receptor; C579167:Chrna7 protein, human; C054634:methyllycaconitine; C004712:cytisine; D000093843:Quinolizidine Alkaloids