TABLE 1.
Benzodiazepine ligand binding to CCK1R and CCK1R-based mutant constructs
NBD, no demonstrable saturable binding; NDB^, no demonstrable saturable binding of BDZ-1 or BDZ-2 radioligands when expressed transiently in COS-1 cells or stably in CHO cells; however, saturable CCK-like radioligand binding confirmed the expression of these constructs on the cell membrane. *, p < 0.05; **, p < 0.001; ***, p < 0.0001 relative to values with wild type CCK1R.
| CCK1R receptor constructs | Abbreviation | CCK1Ra |
|||||
|---|---|---|---|---|---|---|---|
| BDZ-1 radioligand binding, homologous competition Ki | BDZ-1 radioligand binding, heterologous competition Ki | BDZ-2 radioligand binding, homologous competition Ki | BDZ-2 radioligand binding, heterologous competition Ki | BDZ-1 radioligand binding, homologous competition Bmax | BDZ-2 radioligand binding, homologous competition Bmax | ||
| nm | nm | nm | nm | pmol/mg | pmol/mg | ||
| CCK1R-WT | 2.3 ± 0.3 | 197.4 ± 31.7 | NDB | NDB | 19.2 ± 2.5 | NDB | |
| N2.61T | CCK1R TM2 | 3.6 ± 0.7 | 36.2 ± 10.5** | 14.4 ± 1.3*** | 2.4 ± 0.4*** | 61.2 ± 7.2*** | 23.8 ± 1.2*** |
| T3.28V, T3.29S | CCK1R TM3 | 14.5 ± 1.8*** | 259.3 ± 119.1 | NDB | NDB | 33.6 ± 7.5* | NDB |
| I6.51V, F6.52Y | CCK1R TM6 | 10.8 ± 2.2*** | 238.7 ± 29.0 | NDB | NDB | 14.1 ± 0.2 | NDB |
| L7.39H | CCK1R TM7 | 11.4 ± 0.6*** | 104.2 ± 7.9 | NDB | NDB | 46.1 ± 5.3*** | NDB |
| N2.61T + T3.28V, T3.29S | CCK1R TM(2,3) | 17.2 ± 6.7** | 26.6 ± 13.6** | 14.9 ± 1.9*** | 25.5 ± 10.5** | 23.4 ± 6.2 | 38.7 ± 8.3*** |
| N2.61T + I6.51V, F6.52Y | CCK1R TM(2,6) | 32.5 ± 8.7** | 18.1 ± 11.0** | 22.7 ± 7.5** | 23.8 ± 1.9** | 14.8 ± 3.7 | 19.7 ± 1.7*** |
| N2.61T + L7.39H | CCK1R TM(2,7) | 11.0 ± 2.3*** | 4.5 ± 0.2** | 7.1 ± 2.2* | 25.0 ± 4.0** | 33.5 ± 10.0 | 43.4 ± 1.5*** |
| T3.28V, T3.29S + I6.51V, F6.52Y | CCK1R TM(3,6) | NDB^ | NDB^ | NDB^ | NDB^ | NDB^ | NDB^ |
| T3.28V, T3.29S + L7.39H | CCK1R TM(3,7) | NDB^ | NDB^ | NDB^ | NDB^ | NDB^ | NDB^ |
| I6.51V, F6.52Y + L7.39H | CCK1R TM(6,7) | NDB^ | NDB^ | NDB^ | NDB^ | NDB^ | NDB^ |
| N2.61T + T3.28V, T3.29S + I6.51V, F6.52Y | CCK1R TM(2,3,6) | NDB | NDB | 6.4 ± 0.5** | 81.8 ± 4.6 | NDB | 21.1 ± 2.6*** |
| N2.61T + T3.28V, T3.29S + L7.39H | CCK1R TM(2,3,7) | 19.5 ± 1.4*** | 5.1 ± 0.7** | 32.9 ± 4.4*** | 250.3 ± 31.1*** | 20.0 ± 3.6 | 48.1 ± 9.8*** |
| N2.61T + I6.51V, F6.52Y + L7.39H | CCK1R TM(2,6,7) | 36.7 ± 4.7*** | 37.6 ± 9.3* | 37.7 ± 10.1*** | 623.0 ± 67.17*** | 12.4 ± 2.7 | 50.9 ± 15.5*** |
| T3.28V, T3.29S + I6.51V, F6.52Y + L7.39H | CCK1R TM(3,6,7) | NDB^ | NDB^ | NDB^ | NDB^ | NDB^ | NDB^ |
| N2.61T + T3.28V, T3.29S + I6.51V, F6.52Y + L7.39H | CCK1R TM(2,3,6,7) | NDB | NDB | 2.1 ± 0.3 | 86.9 ± 7.1 | NDB | 15.6 ± 1.6*** |
a Values are expressed as means ± S.E.