High-level expression of functional glutamate receptor channels in insect cells

Kari Keinänen (Corresponding Author), Georg Köhr, Peter Seeburg, Marja-Leena Laukkanen, Christian Oker-Blom

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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 alpha-amino-3-hydroxy-5-methylisoxazole-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 GluR-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 postinfection. 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 micrograms of receptor protein per liter of suspension culture should facilitate production of glutamate receptors for biochemical and structural studies.
Original languageEnglish
Pages (from-to)802-806
Number of pages5
JournalBio/Technology
Volume12
Issue number8
Publication statusPublished - 1994
MoE publication typeA1 Journal article-refereed

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Keinänen, K., Köhr, G., Seeburg, P., Laukkanen, M-L., & Oker-Blom, C. (1994). High-level expression of functional glutamate receptor channels in insect cells. Bio/Technology, 12(8), 802-806. https://www.nature.com/articles/nbt0894-802