Figure 1.4.

Profiles of allosteric modulators in various assays and coupling scenarios. (A and B) Potency data for the mGlu₄ PAM VU0400195 in the presence of different concentrations of the orthosteric agonist glutamate are shown for two assay systems. In a G_qi5-mediated calcium assay (A), a maximal concentration of glutamate does not induce the maximal effect possible within the system (E_max(agonist) <E_max(system)), shown as a dotted line for the maximal glutamate alone response. In a different assay system, mGlu₄-mediated activation of G protein inwardly rectifying potassium channels (B), glutamate induces a maximal response alone that cannot be further potentiated by PAMs (E_max(agonist) =E_max(system)); again, dotted line indicated the maximal glutamate response. Data for both assay systems reveal that there is a substantial leftward shift in the potency of the PAM at various glutamate concentrations. In the case of VU0400195, glutamate concentrations that induce no apparent effects alone can be potentiated by this PAM. For example, addition of 330 nM glutamate does not induce an apparent response in either calcium or GIRK assays, yet a concentration-dependent effect is observed in the presence of VU0400195 with a potency of 5–6 μM (white triangles). The presence of increasing concentrations of glutamate results in a dramatic 60-fold left shift in VU0400195 potency (e.g., addition of VU0400195+10 μM glutamate shifts the potency of VU0400195 to 0.1 μM, closed inverted triangles). Therefore, determination of PAM potencies can be dramatically affected by the concentration of orthosteric agonist used in the assay. (C and D). These efficacy experiments again show the ability of a PAM to increase the maximal response in one assay (C), but not in another (D). In both assays, PAM activity can also be observed as a leftward shift of the agonist concentration–response. Data are representative of three independent experiments performed in triplicate (C.M. Niswender, previously unpublished data).