TABLE 4

Comparison of mutation effects on binding and functional potencies of antagonists at OX1

[3H]SB-674042 and [3H]almorexant binding properties at human wild-type and mutated hOX1receptors. Saturation binding isotherms of [3H]SB-674042 and [3H]almorexant were performed on membrane preparations from HEK293 cells transiently transfected with the WT and mutated hOX1 as described under Materials and Methods. The Kd and Bmax values are mean ± S.E., calculated at least from three to five independent experiments (each performed in triplicate). Statistical significance was determined using the two-tailed t test. Effects of mutations on inhibition of orexin-A-induced [Ca2+]i response by SB-674042 and almorexant. Kb and Hill coefficient (nH) values for the inhibition by SB-674042 and almorexant of orexin-A (EC80)-evoked [Ca2+]i response in the HEK293 cells transiently transfected with the hOX1 WT and mutated receptors. Data are means ± S.E. of the 6 dose-response measurements (each performed in duplicate) from three independent transfections. The mutations that affected the binding and functional potencies of [3H]SB-674042 and [3H]almorexant compared with the WT are shown in boldface type.

hOX1Position in the 7TMD[3H]SB-674042 BindingSB-674042 (FLIPR)[3H]Almorexant BindingAlmorexant (FLIPR)
KdKd (mut)/ Kd (WT)BmaxKbKb (mut)/ Kb (WT)nHKdKd (mut)/ Kd (WT)BmaxKbKb (mut)/ Kb (WT)nH
nMpmol/mg proteinnMnMpmol/mg proteinnM
WT1.2 ± 0.014 ± 0.40.6 ± 0.10.5 ± 0.12.3 ± 0.322.9 ± 2.24.8 ± 0.70.5 ± 0.0
Q126A3.3261.1 ± 6.050.9***10.0 ± 3.824.2 ± 8.840.30.9 ± 0.1N.D.B.>10,000
A127T3.3324.2 ± 7.020.2**15.2 ± 3.013.7 ± 1.622.80.5 ± 0.1N.D.B.>10,000
V130A3.362.5 ± 1.22.117.6 ± 0.50.6 ± 0.31.00.7 ± 0.03.4 ± 1.01.513.4 ± 2.313.1 ± 1.92.71.1 ± 0.1
D203A45.510.8 ± 0.10.79.0 ± 0.2N.M.N.D.B.N.M.
W206A45.54N.D.B.N.M.N.D.B.N.M.
Y215A5.38N.D.B.N.M.N.D.B.N.M.
F219A5.42N.D.B.187.0 ± 77.1311.70.5 ± 0.1N.D.B.921.0 ± 346.5191.90.9 ± 0.3
Y224A5.472.2 ± 1.01.82.2 ± 0.20.3 ± 0.00.50.6 ± 0.0N.D.B.22.8 ± 3.74.80.9 ± 0.1
Y311A6.4812.9 ± 3.410.8**2.7 ± 0.30.7 ± 0.11.20.6 ± 0.0N.D.B.>10,000
H344A7.3927.2 ± 4.022.7***1.6 ± 0.812.5 ± 1.120.80.8 ± 0.0N.D.B.294.0 ± 97.161.30.3 ± 0.1
Y348A7.4311.2 ± 1.09.3***3.2 ± 0.14.1 ± 0.66.80.6 ± 0.1N.D.B.157.0 ± 18.832.70.8 ± 0.1
  • N.D.B., no detectable binding due to high nonspecific binding; N.M., not measured.

  • * P < 0.05.

  • ** P < 0.01.

  • *** P < 0.001.