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

ABSTRACT: 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. ?-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 ?1?1?? nAChRs. The Pro?, Gly?, Arg?, 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 ?1?1?? nAChR. On the contrary, substituting Asn?, His10 and Ser12 with Ala respectively did not affect its activity. Interestingly, the [E1A] GI analogue exhibited a three-fold potency for mouse ?1?1?? nAChR, whereas it obviously decreased potency at rat ?9?10 nAChR compared to wildtype GI. Molecular dynamic simulations also suggest that loop2 of GI significantly affects the interaction with ?1?1?? nAChR, and Tyr11 of GI is a critical residue binding with three hydrophobic amino acids of the ? subunit, including Leu93, Tyr95 and Leu103. Our research elucidates the interaction of GI and mouse ?1?1?? nAChR in detail that will help to develop the novel analogues of GI.

SUBMITTER: Ning J

PROVIDER: S-EPMC6315591 | biostudies-literature | 2018 Dec

REPOSITORIES: biostudies-literature

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

Ning Jiong J Li Rui R Ren Jie J Zhangsun Dongting D Zhu Xiaopeng X Wu Yong Y Luo Sulan S

Marine drugs 20181213 12

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 <i>Conus geographus</i> 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. ...[more]

PMID: 30551685

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Project description: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.

Dataset Information

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

Publications

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

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