Figure 4

High affinity Derivatives of SB269652 display lower affinity and cooperatiivty in the absence of a sodium ion coordinated by D80^2.50. (A) In the presence of 100 mM NaCl (closed symbols) the azaindole derivative of SB269652, (4, MIPS1726) acted to partially displace 0.5 nM [³H]spiperone in a competition binding assay using membranes from FlpIN CHO cells stably expressing the WT hD_2LR. These data could be fit to an allosteric ternary complex model to derive a value of affinity and cooperativity with [³H]spiperone (Table 1). In the presence of Na⁺, MIPS1726 displayed lower affinity at the E95^2.65A and D80^2.50 receptor mutants (Table 1). In the absence of Na⁺ (100 mM NMDG, open symbols) MIPS1726 displayed lower affinity at the WT and E95^2.65A D_2LRs but no change in affinity at the D80^2.50A mutant (Table 1). (B) In the presence of 100 mM NaCl (closed symbols) the 7-fluoro-indole-2-carboxamide derivative of SB269652 (5, MIPS1868) acted to partially displace 0.15 nM [³H]spiperone in a competition binding assay using membranes from FlpIN CHO cells stably expressing the WT hD_2LR. These data could be fit to an allosteric ternary complex model to derive a value of affinity for SB269652 and cooperativity with [³H]spiperone (Table 1). In the presence of Na⁺, MIPS1868 displayed lower affinity at the E95^2.65A receptor mutant but lower affinity and cooperativity at the D80^2.50 receptor mutant (Table 1). In the absence of Na⁺ (100 mM NMDG, open symbols) MIPS1868 displayed lower affinity at the WT D_2LR but no change in affinity at the E95^2.65A or D80^2.50A mutants.