High-level expression of functional glutamate receptor channels in insect cells.
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.
Keinänen
K
K
VTT Biotechnology and Food Research, Espoo, Finland.
Köhr
G
G
Seeburg
P H
PH
Laukkanen
M L
ML
Oker-Blom
C
C
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002460:Cell Line; D003902:Detergents / Q000494:pharmacology; D004594:Electrophysiology; D015870:Gene Expression; D018014:Gene Transfer Techniques; D005971:Glutamates / Q000378:metabolism; D018698:Glutamic Acid; D007473:Ion Channels / Q000235:genetics* / Q000502:physiology; D007608:Kainic Acid / Q000378:metabolism; D007700:Kinetics; D009036:Moths / Q000378:metabolism*; D017470:Receptors, Glutamate / Q000235:genetics* / Q000502:physiology; D018350:alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid / Q000378:metabolism
D003902:Detergents; D005971:Glutamates; D007473:Ion Channels; D017470:Receptors, Glutamate; D018698:Glutamic Acid; D018350:alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; D007608:Kainic Acid