Effects of the mutations on [3H]Me-talnetant displacement by [MePhe7]NKB, osanetant, and SB222200 in the membrane preparations from HEK293-EBNA cells transiently expressing WT and mutated hNK3Rs
Ki and nH values for [3H]Me-talnetant binding inhibition by [MePhe7]NKB, osanetant, or SB222200 were calculated as described under Materials and Methods. Values are mean ± S.E. of the Ki calculated from three independent experiments, each performed in duplicate. The mutations that affected the affinity constants of [MePhe7]NKB, osanetant, and SB222200 in comparison with WT are shown in boldface type. Statistical significance was determined using a two-tailed t test.
hNK3R | [MePhe7]NKB | Osanetant | SB222200 | ||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Ki | Ki (Mutant)/Ki (WT) | nH | Ki | Ki (Mutant)/Ki (WT) | nH | Ki | Ki (Mutant)/Ki (WT) | nH | |||||||
| nM | nM | nM | |||||||||||||
| WT | 14.7 ± 2.8 | 0.6 ± 0.0 | 1.0 ± 0.3 | 1.1 ± 0.2 | 5.4 ± 0.5 | 0.9 ± 0.0 | |||||||||
| V95A | 19.4 ± 4.1 | 1.3 | 0.9 ± 0.1 | 14.0 ± 3.9 | 13.6 * | 0.6 ± 0.1 | 27.1 ± 5.4 | 5.0 * | 0.7 ± 0.1 | ||||||
| V95L | 7.9 ± 0.9 | 0.5 | 0.7 ± 0.1 | 1.8 ± 0.4 | 1.8 | 1.0 ± 0.1 | 4.6 ± 0.2 | 0.9 | 0.9 ± 0.0 | ||||||
| V95I/A99S | 12.9 ± 0.6 | 0.9 | 0.8 ± 0.1 | 1.7 ± 0.4 | 1.7 | 0.8 ± 0.1 | 12.5 ± 5.5 | 2.3 | 0.8 ± 0.2 | ||||||
| N138A | 875.0 ± 103.0 | 59.4 *** | 0.9 ± 0.1 | 11.5 ± 0.7 | 11.2 *** | 0.9 ± 0.3 | 21.0 ± 4.9 | 3.9 * | 0.8 ± 0.0 | ||||||
| T139A | 24.7 ± 8.8 | 1.7 | 0.9 ± 0.1 | 0.9 ± 0.7 | 0.9 | 0.6 ± 0.2 | 15.7 ± 2.1 | 2.9 | 0.9 ± 0.0 | ||||||
| V95L/T139A | 7.8 ± 2.9 | 0.5 | 0.6 ± 0.1 | 1.7 ± 0.1 | 1.6 | 1.0 ± 0.0 | 8.9 ± 0.8 | 1.7 | 1.0 ± 0.0 | ||||||
| N142A | > 10,000 | 128.0 ± 4.9 | 124.3 *** | 1.1 ± 0.1 | 0.01 ± 0.001 | 0.002 *** | 0.5 ± 0.0 | ||||||||
| L232A | 147.0 ± 7.8 | 10.0 *** | 0.8 ± 0.1 | 1.5 ± 0.6 | 1.5 | 0.4 ± 0.0 | 15.0 ± 3.8 | 2.8 | 0.8 ± 0.0 | ||||||
| Y315F | 90.8 ± 18.5 | 6.2 * | 1.1 ± 0.1 | 12.6 ± 1.8 | 12.2 ** | 1.1 ± 0.3 | 8.0 ± 0.9 | 1.5 | 0.9 ± 0.1 | ||||||
| S341A | 17.1 ± 3.7 | 1.2 | 0.7 ± 0.1 | 1.2 ± 0.3 | 1.2 | 1.0 ± 0.2 | 5.8 ± 0.5 | 1.1 | 0.9 ± 0.0 | ||||||
| M346A | 207.0 ± 31.8 | 14.1 ** | 1.0 ± 0.1 | 0.9 ± 0.4 | 0.9 | 0.7 ± 0.0 | 10.6 ± 0.6 | 2.0 | 0.7 ± 0.0 | ||||||
| S348A | 27.6 ± 4.4 | 1.9 | 0.8 ± 0.1 | 2.4 ± 1.0 | 2.3 | 0.5 ± 0.1 | 9.2 ± 0.2 | 1.7 | 0.9 ± 0.1 | ||||||