Bacterially expressed human serotonin receptor 3A is functionally reconstituted in proteoliposomes
- Authors
- Na, Jung-Hyun; Shin, Jaeil; Jung, Yuna; Lim, Dongbin; Shin, Yeon-Kyun; Yu, Yeon Gyu
- Issue Date
- Apr-2013
- Publisher
- ACADEMIC PRESS INC ELSEVIER SCIENCE
- Keywords
- Serotonin; Receptor; 5-HT3A; Overexpression; Purification; Reconstitution
- Citation
- PROTEIN EXPRESSION AND PURIFICATION, v.88, no.2, pp.190 - 195
- Journal Title
- PROTEIN EXPRESSION AND PURIFICATION
- Volume
- 88
- Number
- 2
- Start Page
- 190
- End Page
- 195
- URI
- http://scholarworks.bwise.kr/ssu/handle/2018.sw.ssu/11299
- DOI
- 10.1016/j.pep.2013.01.001
- ISSN
- 1046-5928
- 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 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.
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