TABLE 1

[3H]Me-talnetant and [3H]osanetant binding properties at human wild-type and mutated NK3Rs

Saturation binding isotherms of [3H]Me-talnetant and [3H]osanetant were performed on membrane preparations from HEK293-EBNA cells transiently transfected with the WT and mutated hNK3Rs as described under Materials and Methods. The Kd and Bmax values are mean ± S.E., calculated from three independent experiments (each performed in triplicate). The mutations that affected the binding affinities of [3H]Me-talnetant and [3H]osanetant comparison with the WT are shown in bold. Statistical significance was determined using a two-tailed t test.


hNK3R

Position in the 7TMD

[3H]Me-Talnetant Binding

[3H]Osanetant Binding
Kd
Kd (Mutant)/Kd (WT)
Bmax
Kd
Kd (Mutant)/Kd (WT)
Bmax
nM pmol/mg protein nM pmol/mg protein
WT 0.8 ± 0.1 34.3 ± 0.5 0.2 ± 0.0 26.0 ± 0.5
V95A 1.42 3.2 ± 0.1 4.0 *** 46.6 ± 6.6 0.5 ± 0.1 2.3 * 28.6 ± 2.5
V95L 1.42 1.0 ± 0.1 1.2 36.3 ± 12.1 0.3 ± 0.0 1.7 40.9 ± 2.0
V95I/A99S 1.42, 1.46 2.4 ± 0.2 3.0 ** 69.7 ± 11.8 0.7 ± 0.1 3.5 ** 48.3 ± 0.8
M134A 2.53 N.B. N.B. N.B. N.B.
N138A 2.57 2.8 ± 0.5 3.5 ** 45.7 ± 10.5 0.8 ± 0.1 3.8 * 18.8 ± 0.4
T139A 2.58 2.3 ± 0.6 2.8 56.2 ± 11.6 0.3 ± 0.1 1.6 41.1 ± 2.8
V95L/T139A 1.42, 2.58 1.5 ± 0.2 1.9 59.4 ± 5.9 0.2 ± 0.1 1.1 58.3 ± 4.5
N142A 2.61 0.1 ± 0.0 0.1 ** 27.6 ± 0.3 N.B. N.B.
V169M 3.36 N.B. N.B. N.B. N.B.
L232A 45.49 1.4 ± 0.3 1.8 30.1 ± 5.6 0.3 ± 0.0 1.4 15.7 ± 1.0
Y315F 6.51 1.3 ± 0.1 1.7 23.3 ± 1.2 1.8 ± 0.2 9.0 *** 18.6 ± 0.7
S341A 7.38 1.0 ± 0.0 1.3 47.2 ± 2.5 0.1 ± 0.0 0.7 34.2 ± 0.5
F342A 7.39 N.B. N.B. 3.4 ± 0.7 17.0 ** 16.6 ± 2.5
F342M 7.39 N.B. N.B. N.B. N.B.
S341I/F342M 7.38, 7.39 N.B. N.B. N.B. N.B.
M346A 7.43 2.0 ± 0.1 2.5 ** 55.2 ± 2.6 0.2 ± 0.0 1.1 36.1 ± 0.9
S348A
7.45
1.3 ± 0.1
1.7
31.9 ± 1.0
0.3 ± 0.1
1.4
15.2 ± 0.6
  • N.B., no binding.

  • * P < 0.05.

  • ** P < 0.01.

  • *** P < 0.001.