Mutational effect on the potencies of 2-, 3-, and 4-BisPEB and MPEP
BHK cells were transiently transfected with wild-type and mutated mGluR5 using the calcium phosphate method. The intracellular Ca2+ response was measured on an imaging-based plate reader 2 days after transfection. The potency is presented as IC50 and pIC50 (mean ± S.E.M.) and represents data from n independent transfections and experiments (duplicate measurements). Mutants were compared with wild-type mGluR5 (pIC50) by one-way ANOVA combined with Dunnett's multiple comparison test or by a one-sample t test for mutants with IC50 values ≥45 μM.
| 2-BisPEB | 3-BisPEB | 4-BisPEB | MPEP | |||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| IC50 | IC50/IC50 (wt) | pIC50 | n | IC50 | IC50/IC50 (wt) | pIC50 | n | IC50 | IC50/IC50 (wt) | pIC50 | n | IC50 | IC50/IC50 (wt) | pIC50 | n | |
| nM | M | nM | M | nM | M | nM | M | |||||||||
| wt | 79 | 7.10 ± 0.07 | 19 | 2200 | 5.66 ± 0.07 | 19 | 2100 | 5.67 ± 0.06 | 14 | 8.1 | 8.09 ± 0.05 | 19 | ||||
| T632A | 83 | 1.1 | 7.08 ± 0.08 | 3 | 1800 | 0.90 | 5.75 ± 0.03 | 3 | 3500 | 1.7 | 5.45 ± 0.05 | 3 | 4.5 | 0.56 | 8.35 ± 0.02 | 3 |
| T632P | 13 | 0.16 | 7.9 ± 0.3*** | 3 | 790 | 0.35 | 6.1 ± 0.1 | 3 | 2500 | 1.2 | 5.61 ± 0.08 | 3 | 2.0 | 0.25 | 8.7 ± 0.1*** | 3 |
| R648E | 12 | 0.15 | 7.92 ± 0.03*** | 3 | 590 | 0.27 | 6.23 ± 0.08** | 5 | 2000 | 1.0 | 5.7 ± 0.2 | 4 | 0.91 | 0.11 | 9.04 ± 0.06*** | 3 |
| I651F | 3200 | 41 | 5.5 ± 0.1*** | 3 | >45,000 | >20 | —*** | 2 | >45,000 | >21 | —*** | 2 | 500 | 62 | 6.3 ± 0.2*** | 3 |
| P655S | 160 | 2.0 | 6.8 ± 0.1 | 3 | >45,000 | >20 | —*** | 2 | 3200 | 1.5 | 5.5 ± 0.1 | 6 | 60 | 7.4 | 7.22 ± 0.04*** | 3 |
| Y659A | 1100 | 14 | 5.97 ± 0.03*** | 3 | >45,000 | >20 | —*** | 2 | >45,000 | >21 | —*** | 2 | 950 | 120 | 6.02 ± 0.03*** | 3 |
| Y659F | 38 | 0.48 | 7.42 ± 0.01 | 3 | 1200 | 0.54 | 5.93 ± 0.09 | 3 | 630 | 0.30 | 6.2 ± 0.1* | 3 | 5.5 | 0.68 | 8.26 ± 0.09 | 3 |
| N747A | 830 | 11 | 6.08 ± 0.07*** | 3 | >45,000 | >20 | —*** | 2 | 2800 | 1.3 | 5.55 ± 0.08 | 6 | 13 | 1.6 | 7.88 ± 0.06 | 3 |
| N747S | 430 | 5.4 | 6.37 ± 0.01*** | 3 | 25,000 | 11 | 4.6 ± 0.2*** | 3 | 5000 | 2.4 | 5.3 ± 0.1 | 3 | 34 | 4.2 | 7.47 ± 0.06*** | 3 |
| G748L | 79 | 1.0 | 7.1 ± 0.2 | 3 | 4000 | 1.8 | 5.4 ± 0.3 | 3 | 4000 | 1.9 | 5.4 ± 0.1 | 3 | 40 | 4.9 | 7.4 ± 0.2*** | 3 |
| W785A | 890 | 11 | 6.05 ± 0.09*** | 3 | 4300 | 2.0 | 5.37 ± 0.09a | 3 | 6000 | 2.9 | 5.22 ± 0.09* | 3 | 2300 | 280 | 5.64 ± 0.08***,a | 3 |
| F788A | 98 | 1.2 | 7.01 ± 0.02 | 3 | 1900 | 0.86 | 5.72 ± 0.06 | 6 | ∼45,000 | ∼21 | —*** | 2 | 220 | 27 | 6.66 ± 0.06*** | 3 |
| F788W | 260 | 3.3 | 6.59 ± 0.09* | 3 | >45,000 | >20 | —*** | 2 | >45,000 | >21 | —*** | 2 | 79 | 9.8 | 7.1 ± 0.2*** | 3 |
| Y792A | 160 | 2.0 | 6.79 ± 0.03 | 3 | >45,000 | >20 | —*** | 3 | 6300 | 3.0 | 5.20 ± 0.04* | 3 | 66 | 8.1 | 7.18 ± 0.04*** | 3 |
| Y792F | 110 | 1.4 | 6.97 ± 0.04 | 3 | 2500 | 1.1 | 5.6 ± 0.2 | 3 | 1600 | 0.76 | 5.8 ± 0.1 | 3 | 13 | 1.6 | 7.90 ± 0.04 | 3 |
| S809F | 5000 | 63 | 5.3 ± 0.2*** | 3 | 6300 | 2.9 | 5.2 ± 0.2 | 3 | 5000 | 2.4 | 5.3 ± 0.1 | 3 | 4000 | 490 | 5.4 ± 0.1*** | 3 |
| S809A | 1500 | 19 | 5.81 ± 0.03*** | 6 | 2500 | 1.1 | 5.6 ± 0.2 | 3 | 1000 | 0.48 | 6.0 ± 0.2 | 3 | 420 | 52 | 6.38 ± 0.09*** | 5 |
| A810V | 790 | 10 | 6.1 ± 0.1*** | 3 | >45,000 | >20 | —*** | 3 | 7600 | 3.6 | 5.12 ± 0.08** | 3 | 510 | 63 | 6.29 ± 0.04*** | 3 |
| A813F | 120 | 1.5 | 6.91 ± 0.08 | 3 | 3400 | 1.5 | 5.47 ± 0.07 | 3 | 3500 | 1.7 | 5.46 ± 0.03 | 3 | 10 | 1.2 | 7.99 ± 0.06 | 3 |