STABLE EXPRESSION OF RECOMBINANT AMPA RECEPTOR SUBUNITS - BINDING AFFINITIES AND EFFECTS OF ALLOSTERIC MODULATORS

Homomeric AMPA lpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid )-type glutamate receptors (GluRs) were stably expressed in kidney cel ls from cDNAs encoding GluR1 flop, GluR2 flip, GluR2 flop, and GluR3 f lop subunits. The recombinant receptors were of the expected size and showed functional properties in whole-cell recording as previously rep orted. [H-3]AMPA binding to all subunits was increased to a similar ex tent by the chaotropic ion thiocyanate (SCN-). Significant differences were found in the Scatchard plots, however, which were linear and of high affinity for GluR1 and -3 receptors (K-D values of 33 and 52 nM, respectively) but showed curvature for GluR2 receptors, indicating the presence of two components with distinct affinities. As with brain AM PA receptors, solubilization of GluR2 receptors reduced the number of lower-affinity sites and correspondingly increased the number of high er-affinity sites. The sulfhydryl reagent p-chloromercuriphenylsulfoni c acid, which increases binding to brain receptors, produced only mino r changes except in the case of GluR2 flip. These results indicate tha t GluR2, among the subunits examined here, most closely resembles the native AMPA receptors in brain membranes. [3H]AMPA binding was inhibit ed in a noncompetitive manner by two drugs that change the desensitiza tion kinetics of the AMPA receptor. In agreement with physiological ob servations, the apparent affinity of cyclothiazide for GluR2 flip (EC5 0 = 7 mu M) was higher than that for receptors made of flop subunits ( 49-130 mu M) In contrast, BDP-37, a member of the benzamide family of drugs, exhibited a lower potency for GluR2 flip (58 mu M) than for any of the flop isoforms (18-40 mu M). These results predict that the act ion of centrally active AMPA-receptor modulators varies across brain r egions depending on their flip/flop composition.