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High-level Expression of Functional Glutamate Receptor Channels in Insect Cells

Bio/Technology volume 12, pages 802–806 (1994)Cite this article

Abstract

We have expressed glutamate-gated ion channels in Spodoptera frugiperda Sf21 insect cells using a recombinant baculovirus system. Cells infected with recombinant baculoviruses encoding the α-amino-3-hydroxy-5-methyIisoxazole-4-propionate (AMPA)-selective glutamate receptor channel subunits GluR-B and GluR-D displayed specific high-affinity [3H]AMPA binding (apparent dissociation constant Kd of 15 nM for GIuR-B and 40 nM for GluR-D) with pharmacological profiles typical of AMPA receptors. The binding reached maximal levels (Bmax of 15–30 pmol per mg of membrane protein) by 3–4 days post-infection. AMPA, glutamate and kainate triggered inward currents in GluR expressing cells, indicating assembly of functional homomeric channels. Formation of heteromeric GluR-B/D channels in doubly-infected cells was evident from the diagnostic current-voltage relations of AMPA-activated whole-cell currents. For the solubilization of the receptor, nonionic detergents Triton X-100, n-octyl-D-glucoside and n-dodecylmaltoside proved most effective. Detergent-solubilized receptor preparations were stable, retained their characteristic ligand-binding properties and bound to immobilized wheat germ lectin, demonstrating the glycosylation of insect cell-expressed GluR subunits. The expression level of 300–400 μg of receptor protein per liter of suspension culture should facilitate production of glutamate receptors for biochemical and structural studies.

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Authors and Affiliations

  1. VTT Biotechnology and Food Research, P.O. Box 1500, FIN-02044 VTT, Espoo, Finland

    Kari Keinänen & Marja-Leena Laukkanen

  2. Center for Molecular Biology (ZMBH), Heidelberg, D-6900, Heidelberg, Germany

    Georg Köhr & Peter H. Seeburg

  3. Department of Biochemistry and Pharmacy, Åbo Akademi University, FIN-20500, Turku, Finland

    Christian Oker-Blom

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  1. Kari Keinänen
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  2. Georg Köhr
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  4. Marja-Leena Laukkanen
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  5. Christian Oker-Blom
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Correspondence to Kari Keinänen.

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Keinänen, K., Köhr, G., Seeburg, P. et al. High-level Expression of Functional Glutamate Receptor Channels in Insect Cells. Nat Biotechnol 12, 802–806 (1994). https://doi.org/10.1038/nbt0894-802

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