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

• Main Authors: Jiong Ning, Rui Li, Jie Ren, Dongting Zhangsun, Xiaopeng Zhu, Yong Wu, Sulan Luo
• Format: Article
• Language: English
• Published: MDPI AG 2018-12-01
• Series: Marine Drugs
• Subjects: muscle-type nAChRs; α-conotoxin GI; structure-activity relationship; electrophysiological study; molecular dynamic simulations
• Online Access: https://www.mdpi.com/1660-3397/16/12/507
• ISSN: 1660-3397

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. &#945;-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&#8315;activity relationship (SAR) between GI and mouse &#945;1&#946;1&#948;&#949; 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 &#945;1&#946;1&#948;&#949; 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 &#945;1&#946;1&#948;&#949; nAChR, whereas it obviously decreased potency at rat &#945;9&#945;10 nAChR compared to wildtype GI. Molecular dynamic simulations also suggest that loop2 of GI significantly affects the interaction with &#945;1&#946;1&#948;&#949; nAChR, and Tyr¹¹ of GI is a critical residue binding with three hydrophobic amino acids of the &#948; subunit, including Leu⁹³, Tyr⁹⁵ and Leu¹⁰³. Our research elucidates the interaction of GI and mouse &#945;1&#946;1&#948;&#949; nAChR in detail that will help to develop the novel analogues of GI.