3h pirenzepine

192IgG-saporin
(batch 2441969) was acquired
from Millipore (Temecula, CA, USA). RRID AB_94979, [³H]-pirenzepine
(86.0 Ci/mmol, catalog no. NET80250UC), [³H]-oxotremorine
(75.8 Ci/mmol, catalog no. NET671), and [³⁵S]GTPγS
(1250 Ci/mmol, catalog no. NEG030H250UC) were from PerkinElmer (Boston,
MA, USA). Both [³H]- and [¹⁴C]-microscales (catalog
no. ART0123A and catalog no. ARC0146, respectively) were used as standards
in the autoradiographic experiments, ARC (American Radiolabeled Chemicals,
Saint Louis, MO, USA). The β-radiation-sensitive Kodak Biomax
MR films (catalog no. 7358460), bovine serum albumin (BSA) (catalog
no. A4503), carbachol (catalog no. C4382), pirenzepine (catalog no.
P7412), oxotremorine (catalog no. O9126), atropine (catalog no. A0257)
scopolamine (catalog no. S0929), dl-dithiothreitol (DTT)
(catalog no. D5545), adenosine deaminase (ADA) (catalog no. A9876),
guanosine 5′-diphosphate (GDP) (catalog no. G7127), guanosine
5′-O-3-thiotriphosphate (GTPγS) (catalog
no. G8634), ketamine (catalog no. K2753), xylazine (catalog no. X1251),
acetylthiocholine iodide (catalog no. 01480), 2-mercaptobenzothiazole
(MBT) (catalog no. M3302), and tetraisopropyl pyrophosphoramide (iso-OMPA)
(catalog no. T1505) were all acquired from Sigma-Aldrich (St. Louis,
MO, USA).

Cell membranes from FreeStyle 293 cells transiently expressing human M₁R were incubated with T‐495 or MK‐7622 (0.1‐30 μmol/L), ACh (0.003‐3000 μmol/L), and 4 nmol/L [³H]‐pirenzepine (PerkinElmer) in assay buffer (20 mmol/L HEPES, 100 mmol/L NaCl, 10 mmol/L MgCl₂, and 0.1% fatty acid free BSA) for 2 hours at room temperature. The binding was terminated by filtration through GF/C filter plates (PerkinElmer) using a cell harvester (PerkinElmer) and five washed with 300 μL of 50 mmol/L Tris‐HCl. The GF/C plates were dried at 42°C; then, 25 μL of microscint 0 (PerkinElmer) was added. Radioactivity was counted using Topcount (PerkinElmer). Nonspecific binding was defined in the presence of 10 μmol/L atropine. To calculate the cooperativity of a PAM, the [³H]‐pirenzepine binding assay data were fitted to the allosteric ternary complex model,35 using GraphPad Prism 5 software: $Y=\frac{\frac{\left[\mathrm{C}\right]}{K_{\mathrm{C}}}+\frac{{\alpha }_{\text{BC}}[\mathrm{B}][\mathrm{C}]}{K_{\mathrm{B}}{K}_{\mathrm{C}}}}{1+\frac{\left[\mathrm{A}\right]}{K_{\mathrm{A}}}+\frac{\left[\mathrm{B}\right]}{K_{\mathrm{B}}}+\frac{\left[\mathrm{C}\right]}{K_{\mathrm{C}}}+\frac{{\alpha }_{\text{AB}}[\mathrm{A}][\mathrm{B}]}{K_{\mathrm{A}}{K}_{\mathrm{B}}}+\frac{{\alpha }_{\text{BC}}[\mathrm{B}][\mathrm{C}]}{K_{\mathrm{B}}{K}_{\mathrm{C}}}}$
where Y is the fractional specific [³H]‐pirenzepine binding; [A], [B], and [C] are the concentrations of ACh, a PAM, and [³H]‐pirenzepine, respectively; K_A, K_B, and K_C are the equilibrium dissociation constants of ACh, a PAM, and [³H]‐pirenzepine, respectively; and α_AB and α_BC are the cooperativities between a PAM and ACh or [³H]‐pirenzepine, respectively.