Stable expression of homomeric AMPA-selective glutamate receptors in BHK cells

cDNAs encoding glutamate receptor glu(1), glu(2) (Q and R) or glu(4) under control of a constitutively active metallothionine promoter, were transfected into baby hamster kidney cells. Following the addition of selection agent, transfectants expressing high levels of glutamate receptor as measured by [H-3]alpha-amino-3-hydroxy-5-methyl-isoxalazole-4-propionate (AMPA) binding, were selected for further studies. Using glutamate receptor antibodies, the receptor proteins were visualized in Western blotting as having a molecular weight of approximately 100 kDa. [H-3]AMPA binding to the glutamate receptor expressing cell lines revealed that glu(1), glu(2) (Q), and glu(4) receptors displayed a single site in Scatchard analysis with K-d values of 12, 15.7 and 21 nM, respectively. However, the Ca2+ impermeable variant of the glu(2) receptor, glu(2) (R) displayed a curvilinear Scatchard plot. Computer resolution suggested the presence of a high and low affinity state (K-H = 2.9 nM; K-L = 40.7 nM). The pharmacological profile of the [H-3]AMPA binding to these recombinant receptors resembled the high affinity [H-3]AMPA binding site in rat brain showing high affinity for glutamate, quisqualate, and medium affinity for 6-cyano-7-nitro-quinoxaline-2,3-dione, CNQX; 6,7-dinitro-quinoxaline-2,3-dione, DNQX; and 6-nitro-7-sulphanyl-benzo(f)quinoxaline-2,3,dione, NBQX. Kainate displayed low affinity and N-methyl-D-aspartate (NMDA), was inactive in inhibiting specific [H-3]AMPA binding. These cell lines will prove to be important tools in the study of glutamate receptors.