. 2023 May 30;12:e83477. doi: 10.7554/eLife.83477

Table 5. Pharmacological parameters of M₄ muscarinic acetylcholine receptor (mAChR) mutants.

	pERK1/2 interaction assays^*			[³H]-QNB interaction binding assays^†			Study
Constructs	log τ_C ACh ^‡	log τ_C LY298 ^‡	log αβ ^§	pK_i ACh ^¶	pK_B LY298 ^¶	log α ^**
WT M₄ mAChR	2.96 ± 0.14 (4)	1.10 ± 0.09	2.43 ± 0.14	4.51 ± 0.15	4.89 ± 0.12	1.97 ± 0.11	Current/Thal^††
S85A^2.57	3.15 ± 0.11(4)	0.91 ± 0.07	1.75 ± 0.09	4.09 ± 0.11	5.44 ± 0.14	1.43 ± 0.06	Current/Thal
Y89A^2.61	2.53 ± 0.17 (5)	= –3	–0.43 ± 0.27*	5.07 ± 0.45	5.36 ± 0.03	–0.13 ± 0.08 ^{‡ ‡}	Current/Thal
Y92A^2.64	2.26 ± 0.15 (4)	–0.06 ± 0.16*	2.25 ± 0.11	4.15 ± 0.25	4.53 ± 0.15	1.2 ± 0.19 ^{‡ ‡}	Current/Thal
I93T, I94V, K95I	2.57 ± 0.11	2.27 ± 0.19 ^{‡ ‡}	N.T.	4.69 ± 0.11	4.82 ± 0.36	2.14 ± 0.17 ^{‡ ‡}	Nawaratne ^{§ §}
I93T^2.65	2.34 ± 0.09	2.38 ± 0.22 ^{‡ ‡}	N.T.	4.97 ± 0.04	5.36 ± 0.09	2.42 ± 0.16 ^{‡ ‡}	Nawaratne
I94V^2.66	2.34 ± 0.09	1.24 ± 0.09	N.T.	4.71 ± 0.06	5.17 ± 0.08	1.74 ± 0.07	Nawaratne
K95I^2.67	2.00 ± 0.07	0.61 ± 0.09 ^{‡ ‡}	N.T.	4.86 ± 0.05	5.20 ± 0.14	1.24 ± 0.04 ^{‡ ‡}	Nawaratne
Y97A^23.49	2.94 ± 0.11 (4)	2.19 ± 0.07*	3.43 ± 0.12*	4.69 ± 0.17	4.25 ± 0.10 ^{‡ ‡}	2.33 ± 0.12	Current/Thal
W98A^23.50	1.45 ± 0.15* (5)	= –3	2.29 ± 0.10	3.65 ± 0.11 ^{‡ ‡}	4.39 ± 0.04	0.73 ± 0.07 ^{‡ ‡}	Current/Thal
G101A^23.53	2.58 ± 0.09 (4)	0.43 ± 0.08*	2.10 ± 0.07	4.37 ± 0.19	5.03 ± 0.17	1.47 ± 0.01	Current/Thal
D106A^3.26	1.24 ± 0.11	= –3	N.T.	3.95 ± 0.09 ^{‡ ‡}	5.29 ± 0.11	1.51 ± 0.15	Leach ^{¶ ¶}
W108A^3.28	1.49 ± 0.17	= –3	N.T.	4.01 ± 0.06 ^{‡ ‡}	4.24 ± 0.07 ^{‡ ‡}	1.23 ± 0.01 ^{‡ ‡}	Leach
L109A^3.29	1.17 ± 0.14	= –3	N.T.	3.11 ± 0.09 ^{‡ ‡}	4.28 ± 0.14 ^{‡ ‡}	2.54 ± 0.10 ^{‡ ‡}	Leach
D112E^3.32	–0.80 ± 0.16 ^{‡ ‡}	= –3	N.T.	<2	5.56 ± 0.13	0.39 ± 0.11 ^{‡ ‡}	Leach
D112N^3.32	N.D.	N.D.	N.T.	3.19 ± 0.02 ^{‡ ‡}	5.79 ± 0.2 ^{‡ ‡}	0.74 ± 0.08	Leach
Y113A^3.33	N.T.	N.T.	N.T.	2.98 ± 0.12 ^{‡ ‡}	4.97 ± 0.15	0.80 ± 0.10 ^{‡ ‡}	Leach
S116A^3.36	0.82 ± 0.17 ^{‡ ‡}	–0.35 ± 0.45 ^{‡ ‡}	N.T.	3.61 ± 0.10 ^{‡ ‡}	5.12 ± 0.08	1.54 ± 0.05	Leach
N117A^3.37	0.80 ± 0.27 ^{‡ ‡}	–0.27 ± 0.16 ^{‡ ‡}	N.T.	3.64 ± 0.04 ^{‡ ‡}	5.30 ± 0.15	1.57 ± 0.13	Leach
V120A^3.40	1.47 ± 0.11	1.20 ± 0.19	N.T.	5.63 ± 0.05 ^{‡ ‡}	5.41 ± 0.10	1.83 ± 0.11	Leach
D129E^3.49	1.45 ± 0.24	0.78 ± 0.16	N.T.	5.04 ± 0.07	5.59 ± 0.12	1.61 ± 0.16	Leach
D129N^3.49	2.56 ± 0.39	1.86 ± 0.12	N.T.	5.54 ± 0.10 ^{‡ ‡}	5.37 ± 0.20	1.86 ± 0.22	Leach
W164A^4.57	N.D. (3)	= –3	2.17 ± 0.64^***	3.95 ± 0.24	5.15 ± 0.28	ND	Current/Thal
F170A^4.63	3.13 ± 0.17 (5)	2.66 ± 0.12*	3.58 ± 0.17*	4.77 ± 0.2	4.53 ± 0.06	2.23 ± 0.13	Current/Thal
W171A^4.64	2.59 ± 0.17 (5)	1.31 ± 0.11	3.11 ± 0.13	3.91 ± 0.21	4.56 ± 0.15	2.00 ± 0.09	Current/Thal
Q172A^4.65	3.05 ± 0.33 (5)	1.18 ± 0.31	2.71 ± 0.16	4.02 ± 0.09	4.99 ± 0.03	1.54 ± 0.08	Current/Thal
F173A^4.66	3.39 ± 0.11(4)	2.03 ± 0.10*	3.31 ± 0.23*	4.09 ± 0.01	4.78 ± 0.19	1.90 ± 0.14	Current/Thal
Q184A^45.49	3.01 ± 0.10 (4)	1.05 ± 0.08	2.08 ± 0.12	4.25 ± 0.12	5.36 ± 0.04	1.70 ± 0.05	Current/Thal
F186A^45.51	1.99 ± 0.11	N.D.	N.T.	4.85 ± 0.06	NR	NR	Nawaratne
I187A^45.52	2.57 ± 0.10	0.62 ± 0.09	2.07 ± 0.15	3.71 ± 0.12	5.46 ± 0.29	1.07 ± 0.29 ^{‡ ‡}	Current/Thal
Q188A^45.53	2.48 ± 0.15	0.99 ± 0.11	2.35 ± 0.16	4.6 ± 0.22	4.94 ± 0.08	1.49 ± 0.04	Current/Thal
F189A^45.54	2.28 ± 0.11	1.25 ± 0.09	2.67 ± 0.11	4.65 ± 0.02	5.09 ± 0.13	1.99 ± 0.08	Current/Thal
L190A^45.55	2.50 ± 0.14	1.32 ± 0.12	2.81 ± 0.12	4.20 ± 0.06	4.92 ± 0.1	2.06 ± 0.09	Current/Thal
W413A^6.48	0.66 ± 0.12^**, (4)	0.61 ± 0.13^***	3.54 ± 0.09^**,	3.47 ± 0.06 ^{‡ ‡}	4.51 ± 0.37	2.45 ± 0.36	Current/Thal
Y416A^6.51	N.T.	N.T.	N.T.	2.85 ± 0.10 ^{‡ ‡}	NR	NR	Thal
N423A^6.58	3.44 ± 0.15 (3)	0.82 ± 0.10	1.43 ± 0.19*	4.41 ± 0.15	5.02 ± 0.06	1.18 ± 0.08 ^{‡ ‡}	Current/Thal
Q427A^6.62	3.15 ± 0.14 (3)	0.99 ± 0.12	1.64 ± 0.12	4.46 ± 0.03	5.43 ± 0.06	1.36 ± 0.04	Current/Thal
S428P^6.63	1.99 ± 0.09	1.40 ± 0.19	N.T.	5.14 ± 0.03 ^{‡ ‡}	5.17 ± 0.15	1.81 ± 0.11	Nawaratne
D432N^7.32	2.26 ± 0.12	1.25 ± 0.18	N.T.	5.19 ± 0.04 ^{‡ ‡}	5.21 ± 0.2	1.37 ± 0.04	Nawaratne
W435A^7.35	2.58 ± 0.17 (4)	= –3	N.R	3.37 ± 0.08 ^{‡ ‡}	NR	NR	Current/Thal
Y439A^7.39	0.60 ± 0.18 ^{‡ ‡}	N.D.	N.T.	3.33 ± 0.10 ^{‡ ‡}	5.84 ± 0.12	0.49 ± 0.03 ^{‡ ‡}	Nawaratne
W440A^7.40	3.69 ± 0.17^†††(4)	0.84 ± 0.11	1.52 ± 0.20^†††	4.29 ± 0.24	4.94 ± 0.06	0.96 ± 0.04 ^{‡ ‡}	Current/Thal
C442A^7.42	1.49 ± 0.16 ^{‡ ‡}	0.82 ± 0.31	N.T.	4.04 ± 0.07 ^{‡ ‡}	5.35 ± 0.06	1.81 ± 0.03	Nawaratne
Y443A^7.43	0.50 ± 0.16 ^{‡ ‡}	N.D.	N.T.	3.36 ± 0.01 ^{‡ ‡}	6.22 ± 0.05 ^{‡ ‡}	1.16 ± 0.01 ^{‡ ‡}	Nawaratne

Values represent the mean ± SEM from three or more independent experiments with the number of individual experimental replicates from the current study shown in parenthesis.

N.T.: not tested; N.D.: not determined; N.R.: no response; ACh, acetylcholine.

Data and analysis from pERK1/2 assays were generated in the current study, Nawaratne et al, J. Bio. Chem. 2010, and Leach et al, Mol. Pharm. 2011. logτ ACh were calculated from the operational model of agonism. log τ LY298 and log αβ were calculated using a simplified operational model of allosterism.

^†

Data and analysis from [³H]-QNB interaction binding assays were generated in Nawaratne et al, J. Bio. Chem. 2010, Leach et al, Mol. Pharm., and Thal et al, Nature 2016.

^‡

logτ_C = logarithm of the operational efficacy parameter corrected for receptor expression using the maximum number of receptor binding sites as previously determined from Nawaratne et al, J. Bio. Chem. 2010, Leach et al, Mol. Pharm., and Thal et al, Nature 2016.

^§

Logarithm of the functional cooperativity factor between ACh and LY298.

^¶

Negative logarithm of the orthosteric (pK_i) or allosteric (pK_B) equilibrium dissociation constant.

^**

Logarithm of the binding cooperativity factor between ACh and LY298.

^††

Values of logτ_C ACh, log τ_C LY298, and log αβ that were calculated in this study. Other parameters are from Thal et al., 2016.

^{‡ ‡}

Values are significantly different from WT M₄ mAChR as determined in previous studies.

^{§ §}

All values are from Nawaratne et al, J. Bio. Chem. 2010 with logτ corrected for receptor expression.

^{¶ ¶}

All values are from Leach et al, Mol. Pharm. 2011.

^***

Parameters determined from the full Operational Model of Allosterism.

^†††

Values are significantly different from WT M₄ mAChR (p<0.05) calculated by a one-way ANOVA with a Dunnett’s post-hoc test.

Table 5. Pharmacological parameters of M4 muscarinic acetylcholine receptor (mAChR) mutants.

Table 5. Pharmacological parameters of M₄ muscarinic acetylcholine receptor (mAChR) mutants.