Stable expression of human homomeric and heteromeric AMPA receptor subunits in HEK293 cells

Human homomeric and heteromeric alpha-amino-3-hydroxy-5-methyl-4-isoxazolep ropionate (AMPA)-type glutamate receptors (GluRs) were stably expressed in HEK293 cells with cDNAs encoding the flip Splice variant of GluR1. GluR2. G luR3, GluR4 subunit, and the GluR1/GluR2, GluR3/GluR2, and GluR4/GluR2 comb ination. The lethal combination of GluR2 and GluR3 subunits was found in hi gh expression levels of both receptors. The AM PA-evoked current-voltage re lationships demonstrated the functional channel properties, such as a doubl e rectification in GluR1, GluR3, and GluR3 rcceptors, and a linear relation in rcceptors assembled from GluR2 alone and coexpression of GluR2 with the other subunits. All the transfectants exhibited higher selectivity for AMP A than glutamate in dose-dependent current responses. [H-3]AMPA binding rev ealed that the homomeric and heteromeric receptors displayed a single bindi ng site in Scatchard analysis, with dissociation constant (k(d)) values in the range of 14.5-49.3 nM. The B-max values were in the range of 0.57-7.66 pmol/mg protein. The ligand displacement potency for [H-3]AMPA binding was CNQX > glutamate > NS257 in all of the transfectants. These results suggest that stable transformants expressing human homomeric and heteromeric AMPA receptors will be useful tools to define selectivity and potential site of action for AM PA receptor modulators.