tetracycline and cytisine

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.

Topics: Acetylcholine; Aconitine; Alkaloids; alpha7 Nicotinic Acetylcholine Receptor; Azocines; Blotting, Northern; Bungarotoxins; Carbachol; Dimethylphenylpiperazinium Iodide; DNA, Complementary; Gene Expression; Humans; Insecticides; Iodine Radioisotopes; Neuroblastoma; Neurons; Nicotine; Nicotinic Agonists; Plasmids; Protein Synthesis Inhibitors; Quinolizines; Receptors, Nicotinic; Recombinant Proteins; RNA, Messenger; Tetracycline; Transfection; Tubocurarine; Tumor Cells, Cultured; Up-Regulation