Solubilization, purification, and functional reconstitution of 5-hydroxytryptamine3 receptors from N1E-115 neuroblastoma cells.
5-Hydroxytryptamine3 (5-HT3) receptors from N1E-115 neuroblastoma cells were solubilized using 1.1% n-octylglucoside; five other detergents were less effective. Purification was achieved by affinity chromatography using immobilized GR119566X and biospecific elution with quipazine. Saturation analyses with [3H] GR67330 binding revealed high affinity binding to homogeneous populations of sites in both the solubilized (Kd = 0.05 +/- 0.02 nM) and purified (Kd = 0.10 +/- 0.04 nM) preparations. Competition experiments indicated that the solubilized and purified receptor preparations retained the characteristics observed in N1E-115 cells in vivo. Polyacrylamide gel electrophoresis of the purified receptor revealed a single protein band of 54.7 +/- 1.3 kDa. The purified receptor was incorporated into liposomes, and the functional integrity of the protein was demonstrated by measurement of m-chlorophenylbiguanide-stimulated 22Na uptake. Saturation analysis of the reconstituted preparation revealed a Kd of 0.24 +/- 0.07 nM and suggested that 0.2% of 5-HT3 receptors present in the original membrane preparation had been incorporated into liposomes.
Lummis
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SC
MRC Molecular Neurobiology Unit, MRC Centre, Cambridge, UK.
Martin
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D000818:Animals; D001667:Binding, Competitive; D003902:Detergents; D007093:Imidazoles / Q000378:metabolism; D007473:Ion Channels / Q000502:physiology; D007700:Kinetics; D008081:Liposomes; D051379:Mice; D009447:Neuroblastoma / Q000473:pathology / Q000648:ultrastructure*; D011985:Receptors, Serotonin / Q000302:isolation & purification* / Q000378:metabolism / Q000502:physiology; D014407:Tumor Cells, Cultured
D003902:Detergents; D007093:Imidazoles; D007473:Ion Channels; D008081:Liposomes; D011985:Receptors, Serotonin; C065783:GR 67330