Bacterially expressed human serotonin receptor 3A is functionally reconstituted in proteoliposomes
- Bacterially expressed human serotonin receptor 3A is functionally reconstituted in proteoliposomes
- 나정현; 신재일; 정윤아; 임동빈; 신년균; 유연규
- serotonin; receptor; 5-HT3A; overexpression; purification; reconstitution
- Issue Date
- Protein expression and purification
- VOL 88, NO 2, 190-195
- 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 serotonindependent 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 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.
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