Figure 3

Effect of the concentration of [³H]NMS and [³H]QNB on the binding of tacrine to solubilized M₂ receptor from Sf9 cells. (A,B) A schematic representation of inter- and intra-molecular allosteric modulation within an oligomer, shown here as a dimer, one protomer of which is occupied by NMS. Two equivalents of tacrine bind with high affinity and in a cooperative manner to the protomer with a vacant orthosteric site (left panel). One equivalent of tacrine binds with low affinity to the protomer with NMS at the orthosteric site (right panel). In each case, tacrine acts as a negative allosteric modulator of NMS. Tacrine and either [³H]NMS (C,E) or [³H]QNB (D,F) were mixed simultaneously with receptor, and the solution was equilibrated for 21 h ([³H]NMS) or 40 h ([³H]QNB) at 30 °C. The concentrations of each radioligand and the corresponding levels of occupancy in the absence of tacrine were as follows: for [³H]NMS, 3.16 nM and 24.4% (open circle), 17.8 nM and 64.5% (open square), 112 nM and 92% (open triangle); for [³H]QNB, 1.01 nM and 57.4% (filled circle), 1.79 nM and 70.6% (filled square), 10 nM and 93% (filled triangle). Three experiments were performed at each concentration. The data represented in the figure plus similar data acquired at three additional concentrations of [³H]NMS or [³H]QNB were analyzed simultaneously in terms of Eq. (3) (n = 2). The fitted curves are depicted by the lines in the figure, and the parametric values are listed in Table S9.