TABLE 3

Drug binding properties at the EGFP(Δ17)hM1 receptor and its mutants as assessed from equilibrium and kinetic [³H]NMS binding

Equilibrium affinity constants (−log M) are from [³H]NMS saturation studies (pK_d values) and from competition-type experiments analyzed according to competitive (pK_i values) or allosteric ternary complex (pK_X values; cooperativity factor pα) binding models. pEC_50,diss (−log M) and E_max,diss (percentage given in parentheses) parameters refer to midpoint and maximal extent of retardation curves for [³H-NMS] dissociation (Data Analyses). Given are the means ± S.E. for 3–10 separate experiments performed as detailed in Materials and Methods.

	EGFP-wt hM1		EGFP-W405A		EGFP-W405F		EGFP-W400A		EGFP-W400F
	pK_d,_i,xpα	pEC_50,diss (E_max, %)	pK_d,_i,x pα	pEC_50,diss (E_max, %)	pK_d,_i,x pα	pEC_50,diss (E_max, %)	pK_d,_i,x pα	pEC_50,diss (E_max, %)	pK_d,_i,x pα	pEC_50,diss (E_max, %)
[³H]NMS B_max^a	10.02 ± 0.03 445 ± 35^a		10.27 ± 0.04 400 ± 75^a		10.28 ± 0.03 910 ± 125^a		10.04 ± 0.01195 ± 15^a		10.04 ± 0.04 395 ± 45^a
Atropine	9.19 ± 0.03		9.30 ± 0.08		9.57 ± 0.01		9.35 ± 003		9.03 ± 0.06
Pirenzepine	8.03 ± 0.08		7.73 ± 0.01		8.24 ± 0.04		7.65 ± 0.10		7.89 ± 0.23
Carbachol	4.89 ± 0.14		5.58 ± 0.19		5.18 ± 0.01		4.68 ± 0.12		4.53 ± 0.12
Gallamine	5.04 ± 0.06	3.94 ± 0.07 (100)	4.88 ± 0.06	4.00 ± 0.04 (100)	4.74 ± 0.02	3.82 ± 0.05 (100)	3.96 ± 0.03	2.35 ± 0.03 (100)^b	4.10 ± 0.07	3.25 ± 0.07 (80 ± 5)
Gallamine	-1.12 ± 0.02^c	3.94 ± 0.07 (100)	-0.89 ± 0.02^c	4.00 ± 0.04 (100)	-0.78 ± 0.07^c	3.82 ± 0.05 (100)	-1.21 ± 0.04^c	2.35 ± 0.03 (100)^b	-0.92 ± 0.07^c	3.25 ± 0.07 (80 ± 5)
Brucine	4.59 ± 0.02^d	4.49 ± 0.02 (100)	N.A.^e	5.02 ± 0.03 (100)	N.A.^e	4.79 ± 0.03 (100)	N.A.^e	3.31 ± 0.06 (77 ± 4)	N.A.^e	3.68 ± 0.05 (85 ± 3)
	-0.10 ± 0.02^c	4.49 ± 0.02 (100)	N.A.^e	5.02 ± 0.03 (100)	N.A.^e	4.79 ± 0.03 (100)	N.A.^e	3.31 ± 0.06 (77 ± 4)	N.A.^e	3.68 ± 0.05 (85 ± 3)

N.A., not applicable; wt, wild type.
↵a Maximal densities in [³H-NMS] binding sites (B_max, fmol/10⁶ cells) are from saturation experiments.
↵b EC_50,diss is from curve fitting to eq. 8 with E_max,diss constrained to 100%.
↵c Cooperativity factor (negative logarithm).
↵d EC_50,diss was used for curve fitting according to eq. 5 because of nearly neutral cooperativity (Data Analyses).
↵e Not applicable. Over a 1–300 µM concentration range, brucine did not significantly modify equilibrium [³H]NMS binding at the mutants.