Figure 2

The NAM SB269652 has no effect at the hD_2LR in the absence of Na⁺. (A) SB269652 (1) is comprised of a 7-cyano-tetrahydroisoquinoline primary pharmacophore and a indole-2-carboxamide secondary pharmacophore (SP) linked by a cyclohexylene spacer. (B) In the presence of 100 mM NaCl, SB269652 acts to inhibit the binding of both dopamine and [³H]spiperone in a competition binding assay using membranes from FlpIN CHO cells stably expressing the WT hD_2LR. These data could be fitted to an allosteric ternary complex model to derive a value of affinity for SB269652 and values of cooperativity with both dopamine and [³H]spiperone (Table 1). (C) In the absence of sodium ions, but the presence of 100 mM NMDG, no effect of SB269652 upon dopamine or [³H]spiperone affinity at the hD_2LR is observed. (D) In competition binding experiments performed using membranes of FlpIn CHO cells stably expressing a human Myc-hD_2LR and the antagonist [³H]spiperone reveal that in the presence of 100 mM sodium (closed symbols) SB269652 acts to partially displace the radioligand consistent with the action of a negative allosteric modulator (NAM). SB269652 has no effect in the absence of Na⁺ (open symbols) or at the D80^2.50A and E95^2.65A receptor mutants.