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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.

SUBMITTER: Na JH

PROVIDER: S-EPMC4836392 | biostudies-literature | 2013 Apr

REPOSITORIES: biostudies-literature

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Bacterially expressed human serotonin receptor 3A is functionally reconstituted in proteoliposomes.

Na Jung-Hyun JH Shin Jaeil J Jung Yuna Y Lim Dongbin D Shin Yeon-Kyun YK Yu Yeon Gyu YG

Protein expression and purification 20130112 2

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 seroto ...[more]

PMID: 23321066

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Bacterially expressed human serotonin receptor 3A is functionally reconstituted in proteoliposomes.

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Bacterially expressed human serotonin receptor 3A is functionally reconstituted in proteoliposomes.

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