Expression, Biochemistry, and Stabilization with Camel Antibodies of Membrane Proteins: Case Study of the Mouse 5-HT3 Receptor

Hassaı̈ne, Ghérici; Deluz, Cédric; Grasso, Luigino; Wyss, Romain; Hovius, Ruud; Stahlberg, Henning; Tanaka, Takashi; Desmyter, Aline; Moreau, Christophe; Peclinovská, Lucie; Minniberger, Sonja; Mebarki, Lamia; Li, Xiaodan; Vogel, Horst; Nury, Hugues

doi:10.1007/978-1-4939-7151-0_8

Cited by 7 publications

(6 citation statements)

References 23 publications

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“…The wild-type mouse 5-HT3A receptor was expressed in a stable, inducible cell line derived from HEK T-REx 293 cells (ThermoFisher), as previously described 5,30,31 . An inducible cell line was cultured in suspension in flasks in an orbital incubator (typical culture size: 5 liters).…”

Section: Methodsmentioning

confidence: 99%

Conformational transitions of the serotonin 5-HT3 receptor

Polovinkin

Hassaı̈ne

Perot

et al. 2018

Nature

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138

184

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The serotonin 5-HT3 receptor is a pentameric ligand-gated ion channel (pLGIC). It belongs to a large family of receptors that function as allosteric signal transducers across the plasma membrane1,2: upon binding of neurotransmitter molecules to extracellular sites, the receptors undergo complex conformational transitions, which result in transient opening of a pore permeable to ions. 5-HT3 receptors are therapeutic targets for emesis and nausea, irritable bowel syndrome and depression3. Despite the recent accumulation in pLGIC structures4–8, no clear unifying view has emerged on conformational transitions involved in channel gating. Here we report four cryo-EM structures of the full-length mouse 5-HT3 receptor, ranging from 3.2 Å to 4.5 Å resolution, obtained in complex with the anti-emetic drug tropisetron, with serotonin, with serotonin and a positive allosteric modulator. The tropisetron-bound structure resembles those obtained with an inhibitory nanobody5 or without ligand9. The other structures represent new conformations, including that of an open state and of two novel ligand-bound states. We present computational insights into the dynamics of the structures, their pore hydration and free-energy profiles; we characterize motions at the gate level and cation accessibility in the pore. Put together, the data deepen our understanding of the gating mechanism of pLGICs, while capturing ligand binding in unprecedented detail.

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Section: Methodsmentioning

confidence: 99%

Conformational transitions of the serotonin 5-HT3 receptor

Polovinkin

Hassaı̈ne

Perot

et al. 2018

Nature

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138

184

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“…The wild-type mouse 5-HT3A receptor was expressed in a stable and inducible cell line, and purified in the C12E9 detergent as previously described 13,50 .…”

Section: Protein Production and Purificationmentioning

confidence: 99%

The Binding of Palonosetron and Other Antiemetic Drugs to the Serotonin 5-HT3 Receptor

et al. 2020

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Inaccurately perceived as niche drugs, antiemetics are key elements of cancer treatment alleviating the most dreaded side effect of chemotherapy. Serotonin 5-HT3 receptor antagonists are the most commonly prescribed class of drugs to control chemotherapy-induced nausea and vomiting (CINV). These antagonists have been clinically successful drugs since the 1980s, yet our understanding of how they operate at the molecular level has been hampered by the difficulty of obtaining structures of drug-receptor complexes. Here, we report the cryo-EM structure of the palonosetron-bound 5-HT3 receptor. We investigate the binding of palonosetron, granisetron, dolasetron, ondansetron, and cilansetron using molecular dynamics, covering the whole set of antagonists used in the clinical practice. The structural and computational results yield detailed atomic insight into the binding modes of the drugs. In light of our data, we establish a comprehensive framework underlying the inhibition mechanism by the -setron drug family.

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“…The mouse wild-type 5-HT3A receptor was expressed in a previously developed stable, inducible HEK-293 cell line from the parental T-Rex 293 cell line (ATCC CRL-1573) (Hassaine et al, 2013;Hassaïne et al, 2017;Polovinkin et al, 2018). The human wild-type 5-HT3A receptor was also expressed in a developed stable, inducible cell line derived from HEK T-Rex 293 cell line (ATCC CRL-1573).…”

Section: Cultured Cells and Protein Expression For Cryo-emmentioning

confidence: 99%

Structural Determinants for Activity of the Antidepressant Vortioxetine at Human and Rodent 5-HT₃receptors

López-Sánchez,

Munro,

Ladefoged

et al. 2024

Preprint

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SummaryVortioxetine (VTX) is a recent antidepressant that targets a variety of serotonin receptors. We investigate the drug’s molecular mechanism of operation at serotonin 5-HT3receptors (5-HT3R), which features two mysterious properties: VTX acts differently on rodent and human 5-HT3R; VTX appears to suppress any subsequent response to agonists. Using a combination of cryo-EM, electrophysiology, and molecular dynamics, we show that VTX stabilizes a resting inhibited state of the mouse 5-HT3R and an agonist bound-like state of the human 5-HT3R, in line with the functional profile of the drug. We report four human 5-HT3R structures and show that the human receptor transmembrane domain is intrinsically fragile. We also explain the lack of recovery after VTX administration via a membrane partition mechanism.

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Expression, Biochemistry, and Stabilization with Camel Antibodies of Membrane Proteins: Case Study of the Mouse 5-HT3 Receptor

Cited by 7 publications

References 23 publications

Conformational transitions of the serotonin 5-HT3 receptor

Conformational transitions of the serotonin 5-HT3 receptor

The Binding of Palonosetron and Other Antiemetic Drugs to the Serotonin 5-HT3 Receptor

Structural Determinants for Activity of the Antidepressant Vortioxetine at Human and Rodent 5-HT₃receptors

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Expression, Biochemistry, and Stabilization with Camel Antibodies of Membrane Proteins: Case Study of the Mouse 5-HT3 Receptor

Cited by 7 publications

References 23 publications

Conformational transitions of the serotonin 5-HT3 receptor

Conformational transitions of the serotonin 5-HT3 receptor

The Binding of Palonosetron and Other Antiemetic Drugs to the Serotonin 5-HT3 Receptor

Structural Determinants for Activity of the Antidepressant Vortioxetine at Human and Rodent 5-HT3receptors

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Product

Resources

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Structural Determinants for Activity of the Antidepressant Vortioxetine at Human and Rodent 5-HT₃receptors