Fig. 2.
Effect of CX546 on AMPA receptor subunits without and with TARPs. A, HEK293 cells were transiently transfected with the flip variants of GluA1, GluA2, and GluA4. Binding to membranes prepared from these cells was measured using a filtration assay (see under Materials and Methods). Binding was normalized to that without drug; the data are the mean and S.E.M. of 3 (A1), 6 (A2), and 4 (A4) experiments. The data were fitted as described in Fig. 1, and the binding parameters EC50 and Emax are summarized in Table 1. [3H]FW concentrations were 1 nM (A1), 4 nM (A2), and 51 nM (A4), and they were selected to be one-fifth of the published KD value for each subunit (determined at 0°C; Kessler and Arai, 2006); this concentration represents approximately one-tenth of the KD at the temperature used in this study (∼24°C). Average binding without drug was as follows: 0.137 ± 0.03 (A1), 0.275 ± 0.04 (A2), and 0.328 ± 0.03 (A4) pmol/mg protein. B and C, GluA2 or GluA4 was transiently transfected into HEK293 cells alone or in combination with γ3 (A2) or γ4 (A4). The data at each drug concentration are averages of 6 (A2) and 4 (A4) experiments (mean and S.E.M.). Concentration-effect curves with and without TARP differed in each case significantly at p < 0.001 [F(2,10) > 72]. The inset shows that the apparent affinity for FW was reduced slightly by γ3 (KD = 79 nM for A2 plus γ3, versus 50 nM for A2 alone); the reduction was on average 1.5 ± 0.1-fold (n = 3). Therefore, an additional CX546 assay was done with GluA2 plus γ3 at a 1.5-fold increased FW concentration of 6 nM to maintain equidistance from the agonist KD (dashed line, average of 2 experiments). The Emax value at this increased FW concentration was not significantly different from that at 4 nM (125 ± 11 versus 128 ± 9%) (t test, p = 0.9).