TABLE 1.

Binding affinities of N-methylscopolamine and novel selective agonists for M₁ mAChR mutants

The affinity constants pK_d (−log M) for [³H]NMS, NDMC, AC-42, and 77-LH-28-1 are tabulated. A cooperativity factor value α is specified where its inclusion significantly improved the global fit to an allosteric ternary complex model of ligand-[³H]NMS interaction (see Materials and Methods). Otherwise, the interactions seemed competitive. pK_d,NMS values were estimated using three different NMS concentrations, equivalent to 0.1, 2, and 10 times K_d are the means of five different experiments for each, and mutant ± S.E.M. pK_d values for the NSAs are the mean ± S.E.M. of measurements using three different [³H]NMS concentrations (0.1, 2, and 10 times K_d); slope factors of binding curves were close to 1.0.

Helix & Mutant	Ligand pKd (−log M)
	NMS	NDMC	AC42	77-LH-28-1
WT	9.86 ± 0.03	7.41 ± 0.05	6.14 ± 0.12	6.56 ± 0.08
TM2
M79A	9.52 ± 0.12	7.62 ± 0.06	5.43 ± 0.04**	5.78 ± 0.05**
Y82A	9.46 ± 0.04	7.15 ± 0.10	6.25 ± 0.10	6.67 ± 0.20
			α = 0.12	α = 0.046
TM3
L100A	9.72 ± 0.04	7.20 ± 0.03	5.81 ± 0.02	6.71 ± 0.02
W101A	8.90 ± 0.03	6.77 ± 0.06**	8.00 ± 0.06***	9.00 ± 0.10***
				α = 0.018
L102A	9.21 ± 0.01	6.58 ± 0.04**	6.52 ± 0.04**	6.61 ± 0.05
			α = 0.046	α = 0.01
D105E	9.47 ± 0.02	7.04 ± 0.08*	5.86 ± 0.05	6.7 ± 0.05
TM7
Y404A	8.11 ± 0.04	7.01 ± 0.03**	6.03 ± 0.03	6.58 ± 0.03
		α = 0.046
Y404F	8.80 ± 0.008	7.57 ± 0.04	5.89 ± 0.04	6.43 ± 0.05
C407A	8.31 ± 0.04	6.92 ± 0.03**	5.74 ± 0.05*	6.29 ± 0.03
Y408A	8.04 ± 0.03	7.85 ± 0.07**	6.37 ± 0.03	6.37 ± 0.03
Y408F	8.98 ± 0.03	7.49 ± 0.05	5.41 ± 0.04**	5.67 ± 0.03***

^*P < 0.05 significantly different from wild-type receptor.

^**P < 0.01 significantly different from wild-type receptor.

^***P < 0.001 significantly different from wild-type receptor.