Firstly, it was determined whether methanthelinium bromide (MB) binds to human M₁-M₅ (hM₁-hM₅) muscarinic acetylcholine receptors in comparison to the classical muscarinic antagonist N-methylscopolamine (NMS). [³H]NMS dissociation binding experiments revealed an allosteric retardation of dissociation at 100 μM of MB ranging from none in hM₃ to 4.6-fold in hM₂ receptors. Accordingly, global non-linear regression analysis of equilibrium inhibition binding curves between [³H]NMS (0.2 and 2.0 nM) and MB was applied and compared using either an allosteric or a competitive model. The allosteric cooperativity of MB binding within MB/NMS/hM receptor complexes was strongly negative and undistinguishable from a competitive interaction throughout all subtypes. Applying the competitive model to the equilibrium binding data of MB and NMS, suggested competition at all hM subtypes: logK_I (± S.E.) hM₃ = 8.71 ± 0.15, hM₁ = 8.68 ± 0.14, hM₅ = 8.58 ± 0.07, hM₂ = 8.27 ± 0.07 to hM₄ = 8.25 ± 0.11. Secondly, the effects of MB on acetylcholine (ACh) induced hM receptor function showed very strong negative allosteric cooperativity at all subtypes pointing against an allosteric antagonism of MB with ACh. Competition with ACh was characterized by logK_B: hM₁ = 9.53 ± 0.05, hM₄ = 9.33 ± 0.05, hM₅ = 8.80 ± 0.05, hM₂ = 8,79 ± 0.06, to hM₃ = 8.43 ± 0.04. In conclusion, MB, below 1 μM, binds competitively and non-selectively (except for the difference between hM₃ vs. hM₄) to all five hM receptor subtypes with nanomolar affinity and is able to functionally inhibit ACh responses in a competitive fashion, with a slight subtype preference for hM₁ and hM₄.

01 Jun 2016·Journal of Biological ChemistryQ2 · BIOLOGY

Dynamic Regulation of Quaternary Organization of the M1 Muscarinic Receptor by Subtype-selective Antagonist Drugs

Q2 · BIOLOGY

Article

Author: Milligan, Graeme ; Marsango, Sara ; Ward, Richard J ; Godin, Antoine G ; Pediani, John D

Although rhodopsin-like G protein-coupled receptors can exist as both monomers and non-covalently associated dimers/oligomers, the steady-state proportion of each form and whether this is regulated by receptor ligands are unknown. Herein we address these topics for the M1 muscarinic acetylcholine receptor, a key molecular target for novel cognition enhancers, by using spatial intensity distribution analysis. This method can measure fluorescent particle concentration and assess oligomerization states of proteins within defined regions of living cells. Imaging and analysis of the basolateral surface of cells expressing some 50 molecules·μm(-2) human muscarinic M1 receptor identified a ∼75:25 mixture of receptor monomers and dimers/oligomers. Both sustained and shorter term treatment with the selective M1 antagonist pirenzepine resulted in a large shift in the distribution of receptor species to favor the dimeric/oligomeric state. Although sustained treatment with pirenzepine also resulted in marked up-regulation of the receptor, simple mass action effects were not the basis for ligand-induced stabilization of receptor dimers/oligomers. The related antagonist telenzepine also produced stabilization and enrichment of the M1 receptor dimer population, but the receptor subtype non-selective antagonists atropine and N-methylscopolamine did not. In contrast, neither pirenzepine nor telenzepine altered the quaternary organization of the related M3 muscarinic receptor. These data provide unique insights into the selective capacity of receptor ligands to promote and/or stabilize receptor dimers/oligomers and demonstrate that the dynamics of ligand regulation of the quaternary organization of G protein-coupled receptors is markedly more complex than previously appreciated. This may have major implications for receptor function and behavior.

01 Sep 2015·Journal of Biological ChemistryQ2 · BIOLOGY

Structural Insight into Specificity of Interactions between Nonconventional Three-finger Weak Toxin from Naja kaouthia (WTX) and Muscarinic Acetylcholine Receptors

Q2 · BIOLOGY

Article

Author: Arseniev, Alexander S ; Shenkarev, Zakhar O ; Lyukmanova, Ekaterina N ; Dolejsi, Eva ; Dolezal, Vladimir ; Shulepko, Mikhail A ; Chugunov, Anton O ; Utkin, Yuri N ; Tsetlin, Victor I ; Kirpichnikov, Mikhail P ; Janickova, Helena ; Efremov, Roman G ; Dolgikh, Dmitry A ; Paramonov, Alexander S

Weak toxin from Naja kaouthia (WTX) belongs to the group of nonconventional "three-finger" snake neurotoxins. It irreversibly inhibits nicotinic acetylcholine receptors and allosterically interacts with muscarinic acetylcholine receptors (mAChRs). Using site-directed mutagenesis, NMR spectroscopy, and computer modeling, we investigated the recombinant mutant WTX analogue (rWTX) which, compared with the native toxin, has an additional N-terminal methionine residue. In comparison with the wild-type toxin, rWTX demonstrated an altered pharmacological profile, decreased binding of orthosteric antagonist N-methylscopolamine to human M1- and M2-mAChRs, and increased antagonist binding to M3-mAChR. Positively charged arginine residues located in the flexible loop II were found to be crucial for rWTX interactions with all types of mAChR. Computer modeling suggested that the rWTX loop II protrudes to the M1-mAChR allosteric ligand-binding site blocking the entrance to the orthosteric site. In contrast, toxin interacts with M3-mAChR by loop II without penetration into the allosteric site. Data obtained provide new structural insight into the target-specific allosteric regulation of mAChRs by "three-finger" snake neurotoxins.

100 Deals associated with N-Methylscopolamine methylsulfate

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KEGG	Wiki	ATC	Drug Bank
D01976	-	A02B	-

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Indication	Country/Location	Organization	Date
Colic	JP	Alfresa Pharma Corp.	26 Aug 1972

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