TABLE 3

Receptor constructs with combined mutations in o2 and o3/TM7 regions

Affinities (expressed as pKapp) for gallamine and W84 are shown, along with half-times of dissociation of [3H]NMS in the absence of allosteric modulators. To facilitate comparisons, the affinity data have also been transformed to a scale of relative difference between the p Kapp values of the human M2 and M5 receptors (see Materials and Methods). Data shown represent mean ± S.E.M. from three or more assays for pKapp values and six or more assays for half-times. Receptors were expressed in COS-7 cell membranes.

Receptors t½ pKapp Values Relative pKapp
Gallamine W84 Gallamine W84
min mean ± S.E.M. %
M2 4.3 ± 0.4 6.99 ± 0.06 7.66 ± 0.10 100 100
M5 95.2 ± 0.6 5.02 ± 0.04 5.66 ± 0.07 0 0
M2 E172P, Y177Q, N419V, T423H 24.3 ± 1.8 5.24 ± 0.10 5.74 ± 0.11 11.2 4.0
M5 P179E, Q184Y, H478T 32.3 ± 0.9 6.47 ± 0.08 7.86 ± 0.07 73.6 110.0
M5 P179E, Q184Y, V474N, H478T 31.8 ± 1.0 6.36 ± 0.05 7.12 ± 0.14 68.0 73.0
CR3 81.5 ± 4.0 5.26 ± 0.08 6.97 ± 0.12 12.2 69.5
CR3 Y177Q, H478T 32.0 ± 0.7 5.98 ± 0.04 6.05 ± 0.08 48.7 19.5
CR3 V474N 76.7 ± 2.3 5.48 ± 0.06 6.61 ± 0.14 23.4 47.5
CR3 H478T 42.1 ± 3.3 6.34 ± 0.05 7.36 ± 0.06 67.0 85.0
CR3 V474N, H478T 32.0 ± 0.7 6.31 ± 0.06 7.08 ± 0.12 65.5 71.0