TABLE 3

Comparison of mutation effects on binding and functional potencies of antagonists at OX₂

[³H]EMPA and [³H]almorexant binding properties at human wild-type and mutated hOX₂ receptors. Saturation binding isotherms of [³H]EMPA and [³H]almorexant were performed on membrane preparations from HEK293 cells transiently transfected with the WT and mutated hOX₂ as described under Materials and Methods. The K_d and B_max 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 EMPA and almorexant. K_b and Hill coefficient (n_H) values for the inhibition by EMPA and almorexant of orexin-A (EC₈₀ value)-evoked [Ca²⁺]_i response in the HEK293 cells transiently transfected with the hOX₂ WT and mutated receptors. Data are means ± S.E. of the six dose-response measurements (each performed in duplicate) from three independent transfections. The mutations that affected the binding and functional potencies of EMPA and almorexant compared with the WT are shown in boldface type.