Modeling study of mecamylamine block of muscle type acetylcholine receptors

α-Conotoxins (α-CTxs) are small disulfide-rich peptides from venom of Conus species that target nicotinic acetylcholine receptors (nAChRs). The muscle-type nAChRs have been recognized as a potential target for several diseases, such as myogenic disorders, muscle dystrophies, and myasthenia gravis. EI, an α4/7-CTx, mainly blocks α1β1δε nAChRs and has an extra N-terminal extension of three amino acids. In this study, the alanine scanning (Ala-scan) mutagenesis was applied in order to identify key residues of EI for binding with mouse α1β1δε nAChR. The Ala-substituted analogues were tested for their abilities of modulating muscle and neuronal nAChRs in Xenopus laevis oocytes using two-electrode voltage clamp (TEVC) recordings. Electrophysiological results indicated that the vital residues for functional activity of EI were His-7, Pro-8, Met-12, and Pro-15. These changes exhibited a significant decrease in potency of EI against mouse α1β1δε nAChR. Interestingly, replacing the critical serine (Ser) at position 13 with an alanine (Ala) residue resulted in a 2-fold increase in potency at the α1β1δε nAChR, and showed loss of activity on α3β2 and α3β4 nAChRs. Selectivity and potency of [S13A] EI was improved compared with wild-type EI (WT EI). In addition, the structure–activity relationship (SAR) of EI revealed that the “Arg1–Asn2–Hyp3” residues at the N-terminus conferred potency at the muscle-type nAChRs, and the deletion analogue △1–3 EI caused a total loss of activity at the α1β1δε nAChR. Circular dichroism (CD) spectroscopy studies demonstrated that activity loss of truncated analogue △1–3 EI for α1β1δε nAChR is attributed to disturbance of the secondary structure. In this report, an Ala-scan mutagenesis strategy is presented to identify crucial residues that are significantly affecting potency of E1 for mouse α1β1δε nAChR. It may also be important in remodeling of some novel ligands for inhibiting muscle-type nAChRs.

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Nicotine activates cell-signaling pathways through muscle-type and neuronal nicotinic acetylcholine receptors in non-small cell lung cancer cells

Pulmonary Pharmacology & Therapeutics ◽

10.1016/j.pupt.2006.07.001 ◽

2007 ◽

Vol 20 (6) ◽

pp. 629-641 ◽

Cited By ~ 65

Author(s):

Diane L. Carlisle ◽

Xuwan Liu ◽

Toni M. Hopkins ◽

Michelle C. Swick ◽

Rajiv Dhir ◽

...

Keyword(s):

Lung Cancer ◽

Cancer Cells ◽

Nicotinic Acetylcholine Receptors ◽

Acetylcholine Receptors ◽

Small Cell ◽

Neuronal Nicotinic Acetylcholine Receptors ◽

Muscle Type ◽

Small Cell Lung ◽

Nicotinic Acetylcholine ◽

Cell Signaling Pathways

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Potency of nondepolarizing muscle relaxants on muscle-type acetylcholine receptors in denervated mouse skeletal muscle

Acta Pharmacologica Sinica ◽

10.1038/aps.2010.163 ◽

2010 ◽

Vol 31 (12) ◽

pp. 1541-1546 ◽

Cited By ~ 8

Author(s):

Hong Wang ◽

Bin Yang ◽

Guang-wei Han ◽

Shi-tong Li

Keyword(s):

Skeletal Muscle ◽

Acetylcholine Receptors ◽

Muscle Relaxants ◽

Muscle Type ◽

Mouse Skeletal Muscle ◽

Nondepolarizing Muscle Relaxants

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“Weak Toxin” fromNaja kaouthiaIs a Nontoxic Antagonist of α7 and Muscle-type Nicotinic Acetylcholine Receptors

Journal of Biological Chemistry ◽

10.1074/jbc.m100788200 ◽

2001 ◽

Vol 276 (19) ◽

pp. 15810-15815 ◽

Cited By ~ 84

Author(s):

Yuri N. Utkin ◽

Viktoriya V. Kukhtina ◽

Elena V. Kryukova ◽

Florence Chiodini ◽

Daniel Bertrand ◽

...

Keyword(s):

Nicotinic Acetylcholine Receptors ◽

Acetylcholine Receptors ◽

Muscle Type ◽

Nicotinic Acetylcholine

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Ancestral acetylcholine receptor β-subunit forms homopentamers that prime before opening spontaneously

10.1101/2022.01.12.475990 ◽

2022 ◽

Author(s):

Christian J.G. Tessier ◽

R. Michel Sturgeon ◽

Johnathon R. Emlaw ◽

Gregory D. McCluskey ◽

F. Javier Pérez-Areales ◽

...

Keyword(s):

Ion Channels ◽

Acetylcholine Receptor ◽

Acetylcholine Receptors ◽

Common Ancestor ◽

Single Channel ◽

Receptor Activity ◽

Β Subunit ◽

Receptor Function ◽

Muscle Type ◽

Α Subunits

Human adult muscle-type acetylcholine receptors are heteropentameric ion channels formed from two α-subunits, and one each of the β-, δ-, and ϵ- subunits. To form functional channels, the subunits must assemble with one another in a precise stoichiometry and arrangement. Despite being different, the four subunits share a common ancestor that is presumed to have formed homopentamers. The extent to which the properties of the modern-day receptor result from its subunit complexity is unknown. Here we show that a reconstructed ancestral muscle-type β-subunit can form homopentameric ion channels. These homopentamers open spontaneously and display single-channel hallmarks of muscle type acetylcholine receptor activity. Our findings demonstrate that signature features of muscle-type acetylcholine receptor function are independent of agonist, and do not necessitate the complex heteropentameric architecture of the modern-day receptor.

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Alanine-Scanning Mutagenesis of α-Conotoxin GI Reveals the Residues Crucial for Activity at the Muscle Acetylcholine Receptor

Marine Drugs ◽

10.3390/md16120507 ◽

2018 ◽

Vol 16 (12) ◽

pp. 507 ◽

Cited By ~ 7

Author(s):

Jiong Ning ◽

Rui Li ◽

Jie Ren ◽

Dongting Zhangsun ◽

Xiaopeng Zhu ◽

...

Keyword(s):

Nicotinic Acetylcholine Receptors ◽

Acetylcholine Receptors ◽

The Novel ◽

Muscle Type ◽

Alanine Scanning Mutagenesis ◽

Dynamic Simulations ◽

Alanine Scanning ◽

Critical Residue ◽

Hydrophobic Amino Acids ◽

Critical Residues

Recently, the muscle-type nicotinic acetylcholine receptors (nAChRs) have been pursued as a potential target of several diseases, including myogenic disorders, muscle dystrophies and myasthenia gravis, etc. α-conotoxin GI isolated from Conus geographus selectively and potently inhibited the muscle-type nAChRs which can be developed as a tool to study them. Herein, alanine scanning mutagenesis was used to reveal the structure–activity relationship (SAR) between GI and mouse α1β1δε nAChRs. The Pro5, Gly8, Arg9, and Tyr11 were proved to be the critical residues for receptor inhibiting as the alanine (Ala) replacement led to a significant potency loss on mouse α1β1δε nAChR. On the contrary, substituting Asn4, His10 and Ser12 with Ala respectively did not affect its activity. Interestingly, the [E1A] GI analogue exhibited a three-fold potency for mouse α1β1δε nAChR, whereas it obviously decreased potency at rat α9α10 nAChR compared to wildtype GI. Molecular dynamic simulations also suggest that loop2 of GI significantly affects the interaction with α1β1δε nAChR, and Tyr11 of GI is a critical residue binding with three hydrophobic amino acids of the δ subunit, including Leu93, Tyr95 and Leu103. Our research elucidates the interaction of GI and mouse α1β1δε nAChR in detail that will help to develop the novel analogues of GI.

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