Table 2.

Ligand binding properties of representative Cys-substituted mutant M3Rs following Cu-Phen treatment

Receptor	[3H]-NMS binding		Carbachol binding, Ki (μM)
	KD pM	Bmax (pmol/mg protein)
M3′(3C)-Xa	524 ± 98	5.82 ± 1.39	45.7 ± 8.9
K259C	877 ± 365	5.00 ± 2.41	32.7 ± 4.1
R260C	733 ± 137	3.99 ± 1.71	46.1 ± 0.8
E263C	421 ± 210	4.42 ± 0.08	26.0 ± 9.6
L264C	719 ± 96	4.85 ± 0.45	26.2 ± 9.7

Membranes prepared from COS-7 cells expressing the indicated M3′(3C)-Xa-derived Cys-substituted mutant M3R (rat) constructs were incubated with 100 μM Cu-Phen (10 min at 22°C), and radioligand binding assays were performed and analyzed as described in Materials and Methods. Data are shown as means ± se of 2 or 3 independent experiments.