Expanding the Genetic Code in Xenopus laevis Oocytes

Authors

  • Dr. Shixin Ye,

    Corresponding author
    1. Ecole Normale Supérieure, Institut de Biologie de l'ENS, IBENS, 46 rue d'Ulm, Paris 75005 (France)
    2. Inserm, U1024, 46 rue d'Ulm, Paris 75005 (France)
    3. CNRS, UMR8197, 46 rue d'Ulm, Paris 75005 (France)
    • Ecole Normale Supérieure, Institut de Biologie de l'ENS, IBENS, 46 rue d'Ulm, Paris 75005 (France)
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  • Morgane Riou,

    1. Ecole Normale Supérieure, Institut de Biologie de l'ENS, IBENS, 46 rue d'Ulm, Paris 75005 (France)
    2. Inserm, U1024, 46 rue d'Ulm, Paris 75005 (France)
    3. CNRS, UMR8197, 46 rue d'Ulm, Paris 75005 (France)
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  • Stéphanie Carvalho,

    1. Ecole Normale Supérieure, Institut de Biologie de l'ENS, IBENS, 46 rue d'Ulm, Paris 75005 (France)
    2. Inserm, U1024, 46 rue d'Ulm, Paris 75005 (France)
    3. CNRS, UMR8197, 46 rue d'Ulm, Paris 75005 (France)
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  • Dr. Pierre Paoletti

    Corresponding author
    1. Ecole Normale Supérieure, Institut de Biologie de l'ENS, IBENS, 46 rue d'Ulm, Paris 75005 (France)
    2. Inserm, U1024, 46 rue d'Ulm, Paris 75005 (France)
    3. CNRS, UMR8197, 46 rue d'Ulm, Paris 75005 (France)
    • Ecole Normale Supérieure, Institut de Biologie de l'ENS, IBENS, 46 rue d'Ulm, Paris 75005 (France)
    Search for more papers by this author

Abstract

Heterologous expression of ligand-gated ion channels (LGICs) in Xenopus laevis oocytes combined with site-directed mutagenesis has been demonstrated to be a powerful approach to study structure–function relationships. In particular, introducing unnatural amino acids (UAAs) has enabled modifications that are not found in natural proteins. However, the current strategy relies on the technically demanding in vitro synthesis of aminoacylated suppressor tRNA. We report here a general method that circumvents this limitation by utilizing orthogonal aminoacyl-tRNA synthetase (aaRS)/suppressor tRNACUA pairs to genetically encode UAAs in Xenopus oocytes. We show that UAAs inserted in the N-terminal domain of N-methyl-D-aspartate receptors (NMDARs) serve as photo-crosslinkers that lock the receptor in a discrete conformational state in response to UV photo treatment. Our method should be generally applicable to studies of other LGICs in Xenopus oocytes.

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