Na, Jung-Hyun Shin, Jaeil Jung, Yuna Lim, Dongbin Shin, Yeon-Kyun Yu, Yeon Gyu 2024-01-20T12:33:34Z 2024-01-20T12:33:34Z 2021-09-05 2013-04 1046-5928 https://pubs.kist.re.kr/handle/201004/128205 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 Ca2+ ions released from the proteoliposomes. Alanine substitution for Trp178 of 5-HT3A abolished the serotonin-dependent ion-channel activity, confirming the importance of Trpl 78 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. (C) 2013 Elsevier Inc. All rights reserved. English ACADEMIC PRESS INC ELSEVIER SCIENCE LIGAND-BINDING SITE 5-HT3 RECEPTOR ACETYLCHOLINE-RECEPTOR MEMBRANE ISOMERIZATION PURIFICATION PROLINE PROTEIN Bacterially expressed human serotonin receptor 3A is functionally reconstituted in proteoliposomes Article 10.1016/j.pep.2013.01.001 1 PROTEIN EXPRESSION AND PURIFICATION, v.88, no.2, pp.190 - 195 PROTEIN EXPRESSION AND PURIFICATION 88 2 190 195 scie scopus 000316537900003 2-s2.0-84873643442 Biochemical Research Methods Biochemistry & Molecular Biology Biotechnology & Applied Microbiology Biochemistry & Molecular Biology Biotechnology & Applied Microbiology Article LIGAND-BINDING SITE 5-HT3 RECEPTOR ACETYLCHOLINE-RECEPTOR MEMBRANE ISOMERIZATION PURIFICATION PROLINE PROTEIN Serotonin Receptor 5-HT3A Overexpression Purification Reconstitution