Regulation by cycloheximide and lowered temperature of cell-surface alpha7-nicotinic acetylcholine receptor expression on transfected SH-EP1 cells.
Heterologous expression of functional, nicotinic acetylcholine receptors (nAChR) in mammalian cells has been difficult to achieve or optimize, even for nAChR containing only one kind of subunit. In this study, we determined effects of lowered temperature or of exposure to the protein synthesis inhibitor cycloheximide (CHX) on cell surface expression of homomeric alpha7-nAChR in transfected SH-EP1 human epithelial cells. We found that incubation of cells for 2 days at 25 degrees C or in the presence of 0.5-2 microg/mL of CHX caused approximately four- or approximately eight-fold increases, respectively, in surface binding sites for 125I-labeled alpha-bungarotoxin (I-Bgt). These increases were accompanied by increases in peak whole-cell current responses to nicotinic agonists. Either treatment lowered protein synthesis and cell proliferation, but experiments using puromycin indicated that a reduction in protein synthesis or cell proliferation per se was not sufficient to increase surface binding. I-Bgt binding to whole-cell membrane pools increased in response to either treatment, suggesting that the increase in surface binding was due, at least in part, to an increase in intracellular receptor levels. The cyclophilin inhibitor cyclosporin A reduced surface expression in untreated as well as CHX- or 25 degrees C-treated cells. The results suggest practical means for increasing cell surface and functional expression of alpha7-nAChR. Although these effects are not simply due to protein synthesis inhibition or reduced cell proliferation, they do involve an increase in intracellular receptor pool size.
Schroeder
Katherine M
KM
Division of Neurobiology, Barrow Neurological Institute, Phoenix, Arizona 85013, USA.
Wu
Jie
J
Zhao
Lingke
L
Lukas
Ronald J
RJ
D001667:Binding, Competitive / Q000187:drug effects; D002038:Bungarotoxins / Q000493:pharmacokinetics; D002455:Cell Division / Q000187:drug effects / Q000502:physiology; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism*; D003513:Cycloheximide / Q000494:pharmacology*; D006801:Humans; D009447:Neuroblastoma / Q000188:drug therapy* / Q000378:metabolism* / Q000473:pathology; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011978:Receptors, Nicotinic / Q000235:genetics / Q000378:metabolism*; D013696:Temperature; D014162:Transfection; D014407:Tumor Cells, Cultured; D015854:Up-Regulation / Q000187:drug effects; D064569:alpha7 Nicotinic Acetylcholine Receptor
D002038:Bungarotoxins; C579167:Chrna7 protein, human; D011500:Protein Synthesis Inhibitors; D011978:Receptors, Nicotinic; D064569:alpha7 Nicotinic Acetylcholine Receptor; D003513:Cycloheximide