TABLE 1.
Ligand-binding properties of the WT and mutants in the WT background AT1R
The values are presented as the mean ± S.E.M. of at least three independent experiments performed in duplicate. The effect of the mutations on the binding affinity is expressed as ΔKi = Ki(mutant)/Ki(WT-AT1).
| Mutation | Location |
Bmax |
Angiotensin II |
Losartan |
EXP3174 |
Valsartan |
Irbesartan |
|||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
|
Ki |
ΔKi |
Ki |
ΔKi |
Ki |
ΔKi |
Ki |
ΔKi |
Ki |
ΔKi |
|||
| pmol/mg | nM | nM | nM | nM | nM | |||||||
| WT-AT1 | 2.62 ± 0.16 | 0.9 ± 0.2 | 1 | 10.7 ± 1.3 | 1 | 1.0 ± 0.1 | 1 | 2.9 ± 0.3 | 1 | 0.25 ± 0.01 | 1 | |
| V108I | TM3 | 2.68 ± 0.49 | 1.6 ± 0.4 | 1.8 | 1087 ± 165 | 101 | 47.7 ± 5.1 | 47.7 | 316 ± 24.4 | 109 | 23.2 ± 0.8 | 77.3 |
| S109T | TM3 | 1.21 ± 0.49 | 1.9 ± 1.0 | 2.1 | 8649 ± 1654 | 808 | 803 ± 220 | 803 | 5707 ± 1538 | 1968 | 328 ± 52.4 | 1093 |
| Y113A | TM3 | 1.49 ± 0.06 | 12.0 ± 5.2 | 13.6 | 2068 ± 331 | 193 | 384 ± 165 | 384 | 2190 ± 101 | 755 | 62.6 ± 16.9 | 209 |
| A163T | TM4 | 1.04 ± 0.12 | 1.6 ± 0.5 | 1.8 | 120 ± 5.5 | 11.2 | 9.6 ± 1.9 | 9.6 | 20.8 ± 2.6 | 7.2 | 3.1 ± 1.3 | 10.3 |
| E173A | ECL2 | 4.34 ± 0.29 | 2.1 ± 1.0 | 2.3 | 11.6 ± 2.6 | 1.1 | 0.7 ± 0.2 | 0.7 | 2.3 ± 1.0 | 0.8 | 0.18 ± 0.02 | 0.7 |
| F182A | ECL2 | 4.45 ± 0.95 | 17.0 ± 1.3 | 18.3 | 25.1 ± 3.7 | 2.3 | 1.1 ± 0.2 | 1.1 | 6.1 ± 1.2 | 2.1 | 0.30 ± 0.05 | 1 |
| Y184A | ECL2 | 1.53 ± 0.13 | 12.6 ± 0.3 | 14 | 8.8 ± 1.9 | 0.8 | 0.6 ± 0.1 | 0.6 | 2.9 ± 0.03 | 1 | 0.044 ± 0.007 | 0.1 |
| K199A | TM5 | 0.08 ± 0.01 | 19.9 ± 5.8 | 22.1 | 35.4 ± 11.4 | 3.3 | 17.1 ± 2.0 | 17.1 | 214 ± 102 | 73.8 | 0.31 ± 0.06 | 1 |
| K199Q | TM5 | 0.28 ± 0.14 | 16.3 ± 2.8 | 18.1 | 16.8 ± 3.7 | 1.6 | 1.2 ± 0.5 | 1.2 | 10.4 ± 0.1 | 3.6 | 0.24 ± 0.08 | 0.7 |
| H256A | TM6 | 1.63 ± 0.91 | 0.5 ± 0.0 | 0.6 | 11.0 ± 1.8 | 1 | 0.9 ± 0.1 | 0.9 | 3.8 ± 0.1 | 1.3 | 0.24 ± 0.02 | 0.7 |
| Q257A | TM6 | 3.37 ± 0.18 | 1.1 ± 0.4 | 1.2 | 66.9 ± 4.7 | 6.3 | 14.5 ± 2.2 | 14.5 | 69.5 ± 6.4 | 24 | 0.46 ± 0.14 | 1.7 |
| Q257E | TM6 | 2.23 ± 0.36 | 0.38 ± 0.004 | 0.4 | 131 ± 22.4 | 12.2 | 10.5 ± 2.7 | 10.5 | 39.5 ± 3.2 | 13.6 | 2.1 ± 0.9 | 7 |
| Y292A | TM7 | 5.03 ± 0.62 | 1.2 ± 0.1 | 1.3 | 102 ± 18.5 | 9.5 | 3.0 ± 1.9 | 3 | 52.3 ± 6.6 | 18 | 4.3 ± 0.3 | 14.3 |
| N295A | TM7 | 6.04 ± 1.83 | 8.7 ± 0.9 | 9.7 | 1461 ± 143 | 137 | 56.9 ± 12.3 | 56.9 | 227 ± 7.8 | 78.3 | 25.8 ± 4.4 | 86 |
| S109T/A163T | TM3/TM4 | 3.54 ± 0.86 | 1.6 ± 0.1 | 1.8 | 4007 ± 597 | 375 | 290 ± 48.4 | 290 | 2602 ± 89.8 | 897 | 47.4 ± 2.7 | 158 |
| S109T/H256A | TM3/TM6 | 0.41 ± 0.06 | 2.1 ± 0.8 | 2.3 | 11,664 ± 1400 | 1090 | 522 ± 95.0 | 522 | 6651 ± 762 | 2293 | 333 ± 124 | 1110 |
| S109T/N295A | TM3/TM7 | 1.74 ± 0.31 | 2.0 ± 0.6 | 2.2 | 576,050 ± 26,509 | 53,836 | 10,718 ± 247 | 10,718 | <1,000,000 | N.D. | 11,247 ± 776 | 37,490 |
| A163T/H256A | TM4/TM6 | 2.03 ± 0.14 | 2.4 ± 0.1 | 2.7 | 210 ± 19.3 | 19.6 | 9.6 ± 2.4 | 9.6 | 55.8 ± 15.0 | 19.2 | 2.3 ± 0.05 | 7.7 |
| A163T/N295A | TM4/TM7 | 1.46 ± 0.28 | 8.0 ± 2.0 | 8.9 | 7788 ± 1332 | 728 | 254 ± 8.8 | 254 | 740 ± 215 | 255 | 212 ± 7.3 | 707 |
| K199Q/H256A | TM5/TM6 | 0.91 ± 0.08 | 23.0 ± 2.1 | 25.6 | 102 ± 10.5 | 9.5 | 26.8 ± 2.8 | 26.8 | 209 ± 14.4 | 72.1 | 1.5 ± 0.07 | 5 |
| H256A/N295A | TM6/TM7 | 2.98 ± 0.08 | 21.2 ± 1.7 | 23.6 | 191 ± 8.8 | 17.9 | 4.3 ± 0.3 | 4.3 | 30.4 ± 5.9 | 10.5 | 1.3 ± 0.1 | 4.3 |
N.D., not determined.