HIGH-LEVEL EXPRESSION OF FUNCTIONAL GLUTAMATE-RECEPTOR CHANNELS IN INSECT CELLS

We have expressed glutamate-gated ion channels in Spodoptera frugiperd a Sf21 insect cells using a recombinant baculovirus system. Cells infe cted with recombinant baculoviruses encoding the pha-amino-3-hydroxy-5 -methylisoxazole-4-propionate (AMPA)-selective glutamate receptor chan nel subunits GluR-B and GluR-D displayed specific high-affinity [H-3]A MPA binding (apparent dissociation constant K-d of 15 nM for GluR-B an d 40 nM for GluR-D) with pharmacological profiles typical of AMPA rece ptors. The binding reached maximal levels (B-max of 15-30 pmol per mg of membrane protein) by 3-4 days postinfection. AMPA, glutamate and ka inate triggered inward currents in GluR expressing cells, indicating a ssembly of functional homomeric channels. Formation of heteromeric Glu R-B/D channels in doubly-infected cells was evident from the diagnosti c 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. Det ergent-solubilized receptor preparations were stable, retained their c haracteristic 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 mu g of receptor prote in per liter of suspension culture should facilitate production of glu tamate receptors for biochemical and structural studies.