title: Bacterially expressed human serotonin receptor 3A is functionally reconstituted in proteoliposomes.
abstract: Human serotonin receptor 3A (5-HT3A) is a ligand-gated ion channel regulated by serotonin. A fusion protein (P9-5-HT3A) of 5-HT3A with the P9 protein, a major envelope protein of bacteriophage phi6, was highly expressed in the membrane fraction of Escherichia coli, and the expressed protein was purified to homogeneity using an affinity chromatography. P9-5-HT3A was observed as mixed oligomers in detergents. The purified P9-5-HT3A was efficiently reconstituted into proteoliposomes, and the serotonin-dependent ion-channel activity of P9-5-HT3A was observed by measuring the increased fluorescence of Fluo-3 attributed to the formation of a complex with the Ca(2+) ions released from the proteoliposomes. Alanine substitution for Trp178 of 5-HT3A abolished the serotonin-dependent ion-channel activity, confirming the importance of Trp178 as a ligand-binding site. Furthermore, the ion-channel activity of the reconstituted P9-5-HT3A was effectively blocked by treatment with ondansetron, an antagonist of 5-HT3A. The bacterial expression system of human 5-HT3A and the proteoliposomes reconstituted with 5-HT3A would provide biophysical and structural analyses of 5-HT3A.
authors: Department of Chemistry, Kookmin University, Seoul 136-702, Republic of Korea.
authors: Jung-Hyun
authors: JH
authors: Na
authors: Jaeil
authors: J
authors: Shin
authors: Yuna
authors: Y
authors: Jung
authors: Dongbin
authors: D
authors: Lim
authors: Yeon-Kyun
authors: YK
authors: Shin
authors: Yeon Gyu
authors: YG
authors: Yu
mesh_terms: D002118:Calcium / Q000378:metabolism; D002846:Chromatography, Affinity; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D011510:Proteolipids / Q000378:metabolism*; D044406:Receptors, Serotonin, 5-HT3 / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012701:Serotonin / Q000378:metabolism; D015854:Up-Regulation; D014763:Viral Matrix Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D011510:Proteolipids; D044406:Receptors, Serotonin, 5-HT3; D011993:Recombinant Fusion Proteins; D014763:Viral Matrix Proteins; C020485:proteoliposomes; C088019:protein P9, bacteriophage phi6; D012701:Serotonin; D002118:Calcium