TABLE 3.

Ligand binding properties of selected Cys-substituted mutant M3Rs cross-linked at 37 °C

COS-7 cells were transiently transfected with the indicated M3′(3C)-Xa-derived Cys-substituted mutant M3R constructs (4 μg of plasmid DNA each). Radioligand binding assays were performed using membranes prepared from these cells after incubation with CuPhen (100 μm) or CuPhen-free buffer for 10 min at 37 °C. To express the M3′(3C)-Xa construct at different densities, we transfected COS-7 cells with three different amounts of plasmid DNA, as indicated. In all cases, carbachol inhibition binding curves were characterized by Hill coefficients ranging from 0.7 to 0.8. Data are shown as mean ± S.E. of 3–7 independent experiments, each performed in duplicate.

Receptor	[3H]NMS, Bmax		CuPhen buffer
	CuPhen-free buffer	CuPhen buffer	[3H]NMS, KD	Carbachol, Ki
	pmol/mg of protein		nm	μm
Vector	0.01 ± 0.03	0.01 ± 0.00
M3′(3C)-Xa (0.2 μg)	0.45 ± 0.14	0.28 ± 0.08
M3′(3C)-Xa (1 μg)	1.15 ± 0.21	0.99 ± 0.25
M3′(3C)-Xa (4 μg)	3.21 ± 0.40	1.97 ± 0.32	0.44 ± 0.08	33.2 ± 7.0
R176C (i2)	2.44 ± 0.81	0.79 ± 0.33	0.47 ± 0.06	9.0 ± 5.0
T180C (i2)	1.60 ± 0.86	0.31 ± 0.13	1.11 ± 0.29	27.9 ± 3.3
F550C (H8)	1.03 ± 0.27	0.25 ± 0.04	1.15 ± 0.37	19.3 ± 5.3
T553C (H8)	1.66 ± 0.47	0.38 ± 0.07	1.02 ± 0.27	19.7 ± 7.8
T556C (H8)	2.21 ± 0.84	1.26 ± 0.40	0.64 ± 0.12	41.3 ± 12.6