TABLE 1.

Ligand binding and G protein-coupling properties of wild-type and mutant hδORs Ligand- and GTP-binding assays were carried out using membranes that were prepared from stably transfected HEK293_i cells, expressing the wild-type hδOR or the N18Q, N33Q, or N18Q/N33Q mutants. Cells were treated for 24 h with 500 ng/ml tetracycline for the ligand-binding assays or with either 10 ng/ml (hδOR, hδOR(N18Q), hδOR(N33Q)) or 20 ng/ml (hδOR(N18Q/N33Q)) tetracycline for the GTP-binding assays. Saturation binding assays were performed using 0.01–10 nm [³H]diprenorphine to obtain the K_d and B_max values. The GTP-binding assays were carried out using 0.01–1000 nm SNC-80 to obtain the EC₅₀ and E_max values. The receptor densities in membranes used for the GTP-binding assays were measured by a one-point binding assay using 5 nm [³H]diprenorphine, and the values were 2.9 ± 0.6, 2.7 ± 0.5, 2.9 ± 0.3, and 2.6 ± 0.6 pmol/mg protein for the wild-type hδOR, hδOR(N18Q), hδOR(N33Q), and hδOR(N18Q/N33Q), respectively. Analysis of the data was performed using GraphPad Prism. Data represent the mean ± S.E. of three independent experiments, performed in triplicate.

	Ligand binding
Receptor	[3H]Diprenorphine		GTP binding, SNC-80
	Kda	Bmaxb	Basal	Emaxc	EC50d
	nm	pmol/mg protein	cps/10 μg protein	% over basal	nm
hδOR	1.52 ± 0.39	59.5 ± 4.8	3752 ± 539	174 ± 6	12.6 ± 1.8
hδOR(N18Q)	1.34 ± 0.19	41.5 ± 2.4e	3126 ± 75	192 ± 49	18.2 ± 2.6
hδOR(N33Q)	1.57 ± 0.34	36.7 ± 3.0e	3590 ± 357	222 ± 35	21.5 ± 4.2
hδOR(N18Q/N33Q)	1.27 ± 0.38	21.6 ± 3.6e	3044 ± 151	242 ± 17	24.1 ± 3.2f

^aEquilibrium dissociation constant is indicated

^bMaximal specific binding is indicated

^cMaximal stimulation is indicated

^dHalf-maximal effective concentration is indicated

^ep < 0.001

^fp < 0.05 is compared with the wild-type hδOR, using Tukey's post test after analysis of variance test