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

Research output: Contribution to journalArticleScientificpeer-review

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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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Detergents
Propionates
Glutamate Receptors
Insects
Glycosylation
Proteins
Baculoviridae
Ligands
Glutamic Acid
Membranes
Ions
Electric potential
Sf9 Cells
Spodoptera
Wheat Germ Agglutinins
Kainic Acid
Octoxynol
Glucosides
Ion Channels
Suspensions

Cite this

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.
Keinänen, Kari ; Köhr, Georg ; Seeburg, Peter ; Laukkanen, Marja-Leena ; Oker-Blom, Christian. / High-level expression of functional glutamate receptor channels in insect cells. In: Bio/Technology. 1994 ; Vol. 12, No. 8. pp. 802-806.
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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.",
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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, vol. 12, no. 8, pp. 802-806.

High-level expression of functional glutamate receptor channels in insect cells. / Keinänen, Kari (Corresponding Author); Köhr, Georg; Seeburg, Peter; Laukkanen, Marja-Leena; Oker-Blom, Christian.

In: Bio/Technology, Vol. 12, No. 8, 1994, p. 802-806.

Research output: Contribution to journalArticleScientificpeer-review

TY - JOUR

T1 - High-level expression of functional glutamate receptor channels in insect cells

AU - Keinänen, Kari

AU - Köhr, Georg

AU - Seeburg, Peter

AU - Laukkanen, Marja-Leena

AU - Oker-Blom, Christian

N1 - Project code: BEL2031

PY - 1994

Y1 - 1994

N2 - 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.

AB - 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.

M3 - Article

VL - 12

SP - 802

EP - 806

JO - Nature Biotechnology

JF - Nature Biotechnology

SN - 1087-0156

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ER -

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