Identification Of Crucial Residues In α-conotoxin GI,EI Inhibiting Muscle Nicotinic Acetylcholine Receptor And Its Structure-Activity Relationship

nAChRs are composed of five subunits which arrange around a central cation pore and are a member of the ligand-gated ion channel superfamily.The nAChRs are further classified into muscular and neuronal subtypes based on their primary sites of expression.The muscle-type nAChRs are found at the neuromuscular junction,which mediate neuromuscular transmission at the neuromuscular junction（NMJ）.Recently,the muscle-type nicotinic acetylcholine receptors（nAChRs）have been pursued as a potential target of several diseases,including myogenic disorders,muscle dystrophies,myasthenia gravis et al.Conotoxin（CTx）is a kind of active polypeptide secreted by tropical marine mollusks,which can act on different kinds of ion channels and receptors.It can be used as an important tool or probe for pharmacological research.a-conotoxin GI isolated from Conus geographus selectively and potently inhibited the muscle-type nAChRs which can be developed as a tool to study them.Here we used alanine scanning mutagenesis to reveal the structure-activity relationship（SAR）between GI and mouse α1β1δε nAChR.The results indicated the critical residues for inhibiting receptors were Pro⁵,Gly⁸,Arg⁹,and Tyr¹¹ which replacing each of them with alanine（Ala）led to a significant loss in potency at mouse α1β1δε nAChR.On the contrary,substituting Asn4,His¹⁰ and Ser12 with Ala respectively did not affect its activity.Interestingly,the[E1A]GI analogue increased the 3-fold potency for mouse α1β1δε nAChR whereas it obviously decreased potency at neuronal nAChRs compared to wildtype GI.Molecular dynamic simulations also suggest that loop2 of GI significantly affects the interaction withα1β1δε nAChR,and Tyr¹¹ of GI is a critical residue binding with three hydrophobic amino acids of the δ subunit,including Leu93,Tyr95,and Leu103.El is an a4/7-CTx which mainly blocks muscle-type nAChRs and has a three-residue extended N-terminal "tail" beyond the first cysteine（Cys）.In this study,Alanine scanning（Ala-scan）was used as mutagenesis approach to identify key residues from El for binding to mouse α1β1δε nAChR.Additional mutants were also synthesized to probe the functional residues in the N-terminus of a-CTx El.Electrophysiological assays indicated that the key residues for functional activity were His7,Pro⁸,Met12 and Pro¹⁵,due to changes at these positions caused a significant decrease in potency for mouse α1β1δε nAChR.Interestingly,replacing the critical serine（Ser）at position 13 with an alanine（Ala）residue exhibited a 2-fold increase in potency at the α1β1δε nAChR and showed loss of activity on a3β2 and a3(34 nAChRs.Selectivity and potency of[S13A]E1 was improved compared with wild-type EI.Further structure-activity studies of EI revealed that the "Argl-Asn2-Hyp3"residues at the N-terminus conferred potency at the muscle-type nAChRs,and deletion analogue △1-3 EI led to a total loss of activity at the α1β1δε nAChR.CD spectroscopy studies demonstrated that activity loss of truncated analogue △1-3 EI for α1β1δε nAChR is attributed to disturbance of secondary structure.Our research elucidates the interaction of GI,EI and mouse α1β1δε nAChR in detail that will help develop the novel analogues of GI,EI.It may also be important in remodeling of some novel ligands for inhibiting muscle-type nAChRs.