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Article

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

by
Jiong Ning
,
Rui Li
,
Jie Ren
,
Dongting Zhangsun
,
Xiaopeng Zhu
,
Yong Wu
* and
Sulan Luo
*
Key Laboratory of Tropical Biological Resources, Ministry of Education, Key Lab for Marine Drugs of Haikou, Hainan University, Haikou 570228, Hainan, China
*
Authors to whom correspondence should be addressed.
Mar. Drugs 2018, 16(12), 507; https://doi.org/10.3390/md16120507
Submission received: 8 November 2018 / Revised: 25 November 2018 / Accepted: 10 December 2018 / Published: 13 December 2018
(This article belongs to the Special Issue Toxins as Marine-Based Drug Discovery)
Browse Figures
Versions Notes

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. 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.

1. Introduction

Nicotinic acetylcholine receptors (nAChRs) are a member of ligand-gated ion channels that mediate the fast excitatory cholinergic neurotransmission in the central and peripheral nervous system [1,2,3]. In vertebrates, nAChRs are classified into muscle-type and neuronal-type nAChRs based on their primary sites of expression. The muscle-type nAChRs are found at the neuromuscular junction, and they mediate neuromuscular transmission at the neuromuscular junction (NMJ) [4,5]. Noticeably, muscle-type receptors from fetal muscle are composed of a combination of α1β1δγ subunits whereas those from adult muscle have the composition of α1β1δε subunit. Previous research revealed that the muscle-type nAChRs were implicated in pathophysiology conditions, including myasthenia gravis, rhabdomyosarcoma, muscle dystrophies, and muscle atrophy [6,7,8].
Conus is a genus of marine gastropod molluscs which is estimated to have 700 different species distributing in tropical and sub-tropical oceans [9,10]. These gastropods armed with deadly venoms can capture worms, fishes or other mollusks. The venoms are composed of different kinds of bioactive peptides which are named as conotoxins [11,12]. Based on their conserved signal peptide sequences, the various conotoxins are classified into different super families. Further classification into families depends on their patterns of disulfide and pharmacological activities [10,13,14]. Among the conotoxins, α-conotoxins are the most studied and targeted muscle-type or neuronal-type nAChRs with high affinity and selectivity. Typical α-conotoxins are arranged in a CC-Xm-C-Yn-C pattern with two loops and have four cysteine residues to form two disulfide bonds with CysI-CysIII and CysI-CysIV connectivity. The first loop (Xm) contains three or four amino acids (m = 3–4) and the second loop (Yn) consists of three to seven amino acids (n = 3–7) [15,16]. Interestingly, most α-3/5 family conotoxins block muscle-type nAChRs with high potency.
α-conotoxin GI, which was isolated from the Conus geographus venom, is composed of 13 amino acids that selectively inhibited muscle-type nAChRs with the IC50 of 20 nM [17,18]. The structure of GI was firstly determined by two-dimensional NMR [19]. Meanwhile, several studies revealed the binding molecular mechanism between GI and muscle-type nAChRs [20,21]. Hann et al. found the 9-arginine of GI was critical for its high selectivity and activity [22], and Geobe et al. also demonstrated that α-conotoxin [R9A] GI displayed a decrease in affinity for the two acetylcholine-binding sites on Torpedo receptors [23]. However, the role of each amino acid in α-conotoxin GI has remained unidentified [19,24,25]. Nowadays, alanine scanning mutagenesis has become an effective strategy in exploring the relationship between toxins and receptors and has been applied in the α-conotoxins pivotal residue identification [26,27].
In the present study, a series of GI analogues were synthesized and the inhibitory activity on various nAChR subunits were assessed (Figure 1). The results demonstrated that the activities retained for [N4A] GI, [H10A] GI and [S12A] GI compared to GI, whereas their potency decreased nearly more than 10-fold for [P5A] GI, [G8A] GI, [R9A] GI, and [Y11A] GI. More importantly, replacing Glu1 with an Ala led to a three-fold increase in potency at the α1β1δε subtype. Further investigation revealed the interaction between α-conotoxin GI and muscle-type nAChRs. In addition, these analogues have the potential to be developed as a molecular probe for differentiating the subtype of nAChRs [28,29].

2. Results

2.1. Synthesis and Purification of GI and Its Analogues

In this study, standard Fmoc solid phase peptide synthesis strategy was used to synthesize GI and its mutants. A two-step oxidation protocol was used to fold these peptides as described previously [30]. Briefly, CysI and CysIII were protected with S-trityl (S-Trt) while CysII and CysIV were protected with S-acetamidomethyl (S-Acm). The first disulfide bridge between CysI and CysIII was formed using the potassium ferricyanide oxidation method, and then the second disulfide bond was produced through iodine oxidation. All synthesized peptides were monitored by analytical Reversed-Phase High Performance Liquid Chromatography (RP-HPLC), and the purity of each analogue was above 95%. Typically, the retention of the fully oxidized peptide GI and [E1A] GI is 8.82 and 8.57 min respectively, and the molecular weight of GI and [E1A] GI is 1436.50 and 1378.52 Da respectively, which are identical with the theoretical molecular weight (Figure 2). The HPLC chromatogram and ESI-MS (Electrospray ionization mass spectrometry) data of other GI analogues are provided in the Supplementary Materials’ Figures S1–S3.

2.2. Potency of α-Conotoxin GI and Its Analogues at the Mouse α1β1δε nAChR

To better understand the SAR between GI and muscle-type nAChRs, GI and all analogues were firstly tested on mouse α1β1δε nAChR at a single concentration of 10 nM. Figure 3 indicates that the relative amount of inhibitions are evoked by all peptides. Three analogues replacing Asn4, His10 and Ser12 with Ala had little effect on α1β1δε nAChR compared with native GI (67% inhibition). On the contrary, [P5A] GI and [R9A] GI substantially reduced the activity, they exhibited 21.9% and 17.6% inhibition of relative current amplitude. In addition, [G8A] GI and [Y11A] GI analogues resulted in a complete loss of inhibitory activity at the concentration of 10 nM. Notably, only one alanine substitution [E1A] GI showed a significant increase at mouse α1β1δε nAChR versus GI, completely blocking muscle-type nAChR at the concentration of 10 nM.
We also observed that the alanine-substituted analogues affected not only the potency against α1β1δε nAChR but also its current recovery. Meanwhile, Figure 4 illustrates the effects of [Y11A] GI and [E1A] GI on mouse α1β1δε nAChR-mediated current respectively. We could see that GI blockade of mouse α1β1δε nAChR was 75.5% at the concentration of 10 nM, while full blocking of ACh-evoked currents was obtained with 10 nM [E1A] GI. Additionally, the recovery time (50% initial current) was compared between wild peptide GI and mutant [E1A] GI after the blockade by 10 nM toxin. Complete recovery of [E1A] GI was observed within 4 min after α-conotoxin [E1A] GI washout (Figure 4A) while the recovery of GI was accomplished more than 7 min after α-conotoxin GI washout (Figure 4B). [Y11A] GI at the concentration of 10 μM completely inhibited mouse α1β1δε nAChR and the inhibitory ACh-evoked current of [Y11A] GI was fully recovered within 2 min after α-conotoxin [Y11A] GI washout (Figure 4D). However, we noticed that 10 μM GI exhibited higher affinity on heteromeric α1β1δε nAChR, and the inhibitory ACh-evoked current of GI was only completely reversible over 9 min after α-conotoxin GI washout (Figure 4C). Table 1 summarizes the recovery time after blockade by α-conotoxin GI and its analogues.
The concentration–response relationship of GI and its analogues were subsequently assessed on mouse α1β1δε nAChR. Figure 5B and Table 2 summarizes that the four analogues [P5A] GI, [G8A] GI, [R9A] GI and [Y11A] GI reduced the activity significantly and the concentration–response curves of them shifted rightward relative to the native GI. The potencies of [P5A] GI and [R9A] GI were 9.3-fold and 8.5-fold lower than GI. [P5A] GI and [R9A] GI inhibited α1β1δε nAChR with IC50 of 54.72 and 49.79 nM respectively (Figure 5B and Table 2). Furthermore, the substitution of Gly8 and Tyr11 with Ala resulted in potencies more than 20-fold lower than GI. [G8A] GI and [Y11A] GI blocked muscle nAChR with IC50 of 170.60 and 381.20 nM respectively (Figure 5B and Table 2). Strikingly, when we substituted Glu1 with Ala, the potency of [E1A] substantially increased with an IC50 of 1.83 nM (5.85 nM in GI) (Figure 5A and Table 2). The other three mutations, [N4A] GI, [H10A] GI and [S12A] GI preserved similar activity, when compared to wildtype GI (Table 2). Above all, our results demonstrated that a single amino acid substitution in GI had a significant impact on its activity.

2.3. Potency of α-Conotoxin GI and Its Analogues at the Rat Neuronal nAChRs

Next, GI and above critical analogues were tested on other neuronal nAChR subtypes with high concentrations. Glu1, Pro5, Gly8, Arg9 and Tyr11 harboring a substitution to Ala exhibited no activity on α3β2, α3β4, α4β4 and α7 subtypes even up to 10 μM (Figure 6). Interestingly, the inhibition of GI was 50% at the concentration of 10 μM and GI blocked ACh-evoked current of rα9α10 nAChR with an IC50 of 9.35 μM (Figure 7A and Table 3). Moreover, the potencies of [P5A] GI and [G8A] GI increased 2.1-fold and 2.2-fold compared to GI with the IC50 of 4.14 and 4.21 μM respectively (Figure 7 and Table 3). In contrast, replacing Glu1 with Ala led to lower inhibitory activity on rα9α10 nAChR.

2.4. Homology Modeling and Molecular Dynamic Simulation

Molecular models of the interaction between the α1(+)δ(−) binding site and GI were established. The model was refined using molecular dynamics simulations; this was used to illuminate the SAR (Figure 8). According to the model, Glu1 is surrounded by two cysteines (Cys176 and Cys177 in α1 subunit) forming disulfide (Figure 8A). Meanwhile, only one amino acid, Glu147 in the δ subunit was found to act with Glu1, which would produce electrostatic repulsion. When Glu1 was substituted by Ala, the repulsive force between them decreased, contributing to a three-fold increased blockade of α1β1δε nAChR. Among all analogues, [Y11A] GI had the highest decrease at the potency on α1β1δε nAChR. Through 40 ns dynamic stimulation, Tyr11 is impacted by more amino acids in the δ subunit, including Leu93, Tyr95 and Leu103 (Figure 8B) forming a relatively hydrophobic environment. Replacing polar residue Tyr11 with a hydrophobic Ala significantly perturbed the affinity potency of GI to the δ subunit, explaining the activity loss of [Y11A] GI to α1β1δε nAChR.

3. Discussions

Until now, several conotoxins have been reported as muscle-type nAChRs inhibiters. The selected conotoxins from different conus species that targeted muscle-type nAChRs are summarized in Table 4. So far, five different families targeting the muscle-type nAChRs have been identified, including α-conotoxins, αB-conotoxins, αD-conotoxins, αO-conotoxins and αS-conotoxins, among which α-3/5 conotoxins become dominant with their high affinity and selectivity. Some conotoxins of other families also inhibited muscle nAChRs but with less selectivity. For example, αB-VxXXIVA cloned from Conus vexillum inhibited mouse α1β1δε nAChR and rat α9α10 nAChR with the IC50 of 6.6 μM and 1.2 μM, respectively [31,32]. Another conotoxin cloned from Conus generalis, named αO-GeXVIA, blocked various nAChR subtypes with nanomolar potency, including rat α9α10 nAChR and mouse α1β1δε nAChR [33]. Moreover, two other conotoxins also cloned from Conus generalis, named αD-GeXXA and αO-GeXXVIIA, blocked muscle-type nAChR and neuronal nAChRs [34,35].
The fetal muscle-type nAChRs contain two ligand binding sites located at the extracellular domain, α/γ or α/δ interface. The specific binding sites of these conotoxins have aroused attention, and several conotoxins were used to investigate the binding affinity on the α/γ or α/δ interface [20,36]. Firstly, three α-conotoxins, GI, MI and SIA conferred 10,000-fold greater affinity at the α/δ versus α/γ interface of muscle nAChR on BC3H-1 cells [20,37,38]. On the contrary, they displayed a higher affinity at the α/γ interface for Torpedo nAChR [39,40,41]. Hann et al. revealed that GI and MI exhibited similar physiological potencies on mouse nAChR while α-conotoxin SI was much less effective than them [21]. In addition, SI failed to discriminate between the α/γ and α/δ interface on mouse and Torpedo nAChR compared with GI or MI [21]. Previous investigation revealed that α-AC1.1b(CIA) from Conus achatinus strongly preferred the α/δ interface instead of the α/γ binding site on the mouse muscle nicotinic acetylcholine receptor [42]. Recently, Giribaldi et al. revealed that CIA also blocked the neuronal rat α3β2 nAChR with micromolar potency [43]. As we know, α-4/7-conotoxin was generally regarded as targeting various neuronal nAChRs, however, one α-4/7-conotoxin EI isolated from Conus ermineus was identified as selectively targeting the α/δ interface of the Torpedo muscle-type nAChRs [44], another α-4/7-conotoxin Lo1a has been characterized to differentiate between muscle-type nAChRs and neuronal-type nAChRs, and this conotoxin had a higher affinity at the α/ε interface [45]. Interestingly, αA-conotoxin OIVB with six cysteines isolated from Conus obscurus venoms from Indo-Pacific, exhibited 1800-fold lower affinity for adult muscle nAChRs, and this toxin preferentially bound to the α/γ interface [46]. Whereas ψ-conotoxin PrIIIE, with CysI–CysIV, CysII–CysV, and CysIII–CysVI disulfide configuration displays higher potency against the adult subtype than the fetal subtype nAChR [47,48].
The structure of GI was revealed by NMR spectroscopy and X-ray crystallography [19,24,49]. A right-handed helical turn containing an Asn4-Cys7 β-turn in the Gly8 to Tyr11 region is a typical structural feature of GI [24]. Gray et al. assumed that the shape of peptide GI was a triangular slab with Glu1, Pro5 and Arg9 situated at the corner [24]. As we know, the proline of loop1 in many α-conotoxins is conservative, and this amino acid plays a crucial role in 310-helix turn forming of conotoxins. Furthermore, Dutertre et al. demonstrated that 310-helix turn might play a crucial role in defining both the ligand conformation and receptor-binding activity [50]. Olivera, B. M el al. found that substituting Pro with Ala in α-conotoxin MI dramatically reduced the toxin’s potency at the α/δ site [51]. Similarly, the Pro5 mutation in GI might alter β-turn secondary structures, significantly reducing the activity of α1β1δε nAChR. Moreover, when we replaced Gly8, Arg9 and Tyr11 with Ala, three analogues, [G8A] GI, [R9A] GI and [Y11A] GI, suffered respectively 29.1-fold, 8.5-fold and 65.1-fold loss of their potencies on α1β1δε nAChR compared to GI, suggesting that the substitution of Gly8, Arg9 and Tyr11 with Ala probably had a significant impact on the right-handed helical turn. Meanwhile, Tyr11 in α-conotoxin GI was located at a general hydrophobic pocket by molecular modeling, and we also noticed that it interacts with hydrophobic amino acids, including Leu93, Tyr95 and Leu103 in the δ subunit (Figure 8B), which is consistent with previous studies that showed α-conotoxin MI interacted strongly with the δ subunit [40,41]. Janes, R. W et al. also suggested that a hydrophobic phenylalanine in this position contributed to anchoring to the receptor [52]. In contrast to the loss of inhibitory activity at most alanine substitutions, Glu1 replaced by Ala in α-conotoxin GI substantially improved the functional activity at α1β1δε nAChR.
In addition, we observed a different current recovery time after the blockade by α-conotoxin GI, [E1A] GI and [Y11A] GI at the mouse α1β1δε nAChR, suggesting that single amino acid mutation possibly changed the interaction between ligands and receptors. Further research is needed in order to elucidate the molecular mechanism underlying their interaction. Various neuronal nAChRs, including α3β2, α3β4, α4β4, α7, and α9α10 were also screened. The electrophysiology assay indicated that alanine-substituted analogues failed to inhibit these receptors, except for α9α10. GI, [P5A] GI and [G8A]GI retained their activity at α9α10 at the micromole level. In contrast, [E1A] GI had little influence on α9α10 nAChR at a high concentration, and the selectivity of [E1A] GI at muscle α1β1δε versus neuronal α9α10 nAChR was improved compared to wildtype GI.

4. Materials and Methods

4.1. Peptide Synthesis and Oxidative Folding of α-Conotoxin GI and Its Analogues

All the α-conotoxin GI and its analogues’ linear peptides were successfully synthesized using Fmoc chemistry and standard side protection, except for four cysteines [30]. The cysteine residues were orthogonally protected using the acid liable S-trityl groups and the acid-stable S-acetomedomethyl groups. After cleavage of the assembled peptide chain from the resin, the first disulfide bond (the S-trityl groups on CysI and CysIII) in each peptide was formed by incubating the peptides in 5 mM ferricyanide (pH 7.5, 0.2 mg/mL) 45 min at 25 °C. The second disulfide bond (S-acetomedomethyl groups on CysII and CysIV) was formed by incubating peptides in 0.4 mM I2 (0.4 mg/mL) 1% TFA under nitrogen protection conditions for 10 min. Then the reaction was quenched by adding 1 M ascorbic acid until the mixture became colorless. The peptide was purified by preparative RP-HPLC. Analytical RP-HPLC and electrospray-mass spectroscopy (ESI-MS) confirmed the purity and molecular mass of oxidized peptides.

4.2. Peptide Quantification

The concentration of purified α-conotoxin GI and its analogues was quantified using an absorbance measurement with a spectrophotometer at a wavelength of 280 nm, calculated with the Lambert–Beer equation, A = εcl. Where A is the absorbance, ε is the extinction coefficient, l is the cuvette path length, c indicates the concentration, and ε is determined with the peptide properties calculator.

4.3. cRNA Preparation and Injection into Xenopus laevis Oocytes

Capped RNA (cRNA) for the various subunits were prepared using the mMESSAGE mMACHINE in vitro transcription Kit (Ambion, Austin, TX, USA) following linearization of the plasmid. The cRNA was purified using MEGA clearTm Transcription Clean-up Kit (Ambion, Austin, TX, USA). The concentration of each cRNA was confirmed by Smart SpecTM plus Spectrophotometer (Bio-Rad, Hercules, CA, USA), with the absorbance monitored at 260 nm. Oocytes were extracted from mature female Xenopus laevis as described previously [30]. cRNAs of mouse α1, β1, δ, ε subunits were mixed at 2:1:1:1 ratio with the final concentration of approximately 50 ng/μL for each subunit cRNA. 50.6 nL of this mixture was injected into each Xenopus lavies oocyte using a Drummond microdispenser (Drummond Scientific, Broomall, PA, USA), and then were incubated at 17 °C in ND96 buffer (96.0 mM NaCl, 2.0 mM KCl, 1.8 mM CaCl2, 1.0 mM MgCl2, 5 mM HEPES, pH 7.1–7.5) supplemented with 10 μg/mL of penicillin, 10 μg/mL of streptomycin and 100 μg/mL of gentamicin before recording. Oocytes were injected within 1 day of harvesting and recordings were carried out 2–7 days after microinjection.

4.4. Two-Electrode Voltage Clamp Electrophysiological Recordings of nAChRs Expressed in Xenopus laevis Oocytes

Two-electrode voltage-clamp recordings from oocytes were carried out at room temperature using an Axonclamp 900A amplifier (molecular devices crop., Sunnyvale, CA, USA). The voltage-recording and current-injecting electrodes were pulled with borosilicate glass and had a resistance of 5–50 megaohms when supplemented with 3 M KCl. The concentration of ACh was 10 μM trials with α1β1δε and α9α10 subtypes, 200 μM trials with α7, and 100 μM for all other subtypes. Oocytes were exposed to a 50 μL cylindrical oocyte recording chamber fabricated from Sylgard, and it was gravity-perfused with ND96 buffer at a rate of ~2 mL/min. All toxin solutions also contained 0.1 mg/mL bovine serum albumin to reduce nonspecific adsorption of the peptide. During recording, the oocytes were clamped at a holding potential of −70 mV. Oocytes were gravity-perfused with standard ND96 solution and supplied once per minute with one second ACh pulse. As a stable baseline was achieved, either ND96 alone or ND96 containing varying concentrations of GI and its analogues were perfusion-applied in a static bath for 5 min before the agonist ACh was added. The electrophysiology data were recorded and analyzed using Clampfit 10.2 software (Molecular Devices Corp., Sunnyvale, CA, USA).

4.5. Data Analysis

The effects of α-conotoxin GI and its analogues on ACh-evoked nAChR-mediated currents were defined as peak current amplitudes relative to the average peak current amplitudes of three control ACh applications, and this was used to normalize the amplitude of each test response to obtain a “% response” or “% block”. The concentration–response curves were fit to the pooled data by Equation (1).
% response = 100/{1 + ([toxin]/IC50)nH}.
where nH is the slope factor (Hill slope) and IC50 is the peptide concentration that gave 50% inhibition of the maximal response. All the electrophysiological data were statistically analyzed using GraphPad Prism 6, with significant differences between the control GI and the analogues determined by t-test.

4.6. Molecular Modeling, Docking, and Dynamic Simulation

We resolve to clarify the molecular mechanism of GI and its analogues acting with muscle-type nAChRs. To begin, we constructed homology models of α1 and δ subunit with the program Modeler 9v10. Nicotinic acetylcholine receptor (code: 2BG9) from Torpedo was adopted as the template [53], and the interface was formed using Lymnaea stagnalis acetylcholine-binding protein (Ls-AChBP; PDB code: 1I9B) as the template [54]. Then the models were optimized with energy minimized by 100 ps of a standard molecular dynamic process with Gromacs 5.1. The structures of GI and its analog [Y11A] GI were used to dock to the α1δ nAChR interface using the program FlexpepDock. All dynamic simulations were performed using Gromacs 5.1. Each dynamic simulation was performed for 40 ns. The interatomic contact difference plot was calculated by determining the total number of toxin contacts with 4 Å of each receptor residue.

5. Conclusions

In summary, we identified SAR between GI and the mouse α1β1δε nAChRs using an alanine-scan strategy. To be specific, peptide [E1A] GI selectively inhibited mouse α1β1δε nAChRs with better potency than native GI. Furthermore, [E1A] GI reduced the potency of neuronal α9α10 nAChRs subtype and improved its selectivity on mouse muscle nAChRs. Compared to wildtype GI, four peptides i.e., [P5A] GI, [G8A] GI, [R9A] GI and [Y11A] GI displayed a dramatic loss of activity at the mouse α1β1δε nAChR. Meanwhile, molecular dynamic simulations demonstrate a relatively hydrophobic Tyr11 of GI, a critical residue, binding with the δ subunit at the mouse α1β1δε nAChR. For neuronal nAChRs, only two variants, [P5A] GI and [G8A] GI, have a slight increase at the α9α10 nAChR versus GI. Taken together, our work expanded our knowledge on SAR between GI and the muscle-type nAChRs, providing sufficient data for the redesign of GI analogues with higher affinity and selectivity.

Supplementary Materials

The following are available online at https://www.mdpi.com/1660-3397/16/12/507/s1, Figure S1: The HPLC chromatograms and mass spectrum of GI, [E1A] GI and [N4A] GI respectively; Figure S2: The HPLC chromatograms and mass spectrum of [P5A] GI, [G8A] GI and [R9A] GI respectively; Figure S3: The HPLC chromatograms and mass spectrum of [H10A] GI, [Y11A] GI and [S12A] GI respectively.

Author Contributions

J.N., Y.W., and S.L. conceived and designed the experiments; J.N., Y.W. and R.L. performed the experiments; J.R. and Y.W. verified this experiment; J.N., X.Z., J.R., Y.W., D.Z. and S.L. analyzed the data; J.N., Y.W. and S.L. wrote the paper.

Funding

This research was funded by National Natural Science Foundation of China (No. 81660585, No. 31760249 and No. 81872794), Major International Joint Research Project of National Natural Science Foundation of China (81420108028), Hainan University Youth foundation (hdkyx201725), Changjiang Scholars and Innovative Research Team in University Grant (IRT_15R15).

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Hurst, R.; Rollema, H.; Bertrand, D. Nicotinic acetylcholine receptors: From basic science to therapeutics. Pharmacol. Therapeut. 2013, 137, 22–54. [Google Scholar] [CrossRef] [PubMed]
  2. Lebbe, E.K.; Peigneur, S.; Wijesekara, I.; Tytgat, J. Conotoxins Targeting Nicitinic Acetylcholine Receptor: An Overview. Mar. Drugs 2014, 12, 2970–3004. [Google Scholar] [CrossRef] [PubMed]
  3. Mir, R.; Karim, S.; Kamal, M.A.; Wilson, C.M.; Mirza, Z. Conotoxins: Structure, Therapeutic Potential and Pharmacological Applications. Curr. Pharm. Des. 2016, 22, 582–589. [Google Scholar] [CrossRef] [PubMed]
  4. Kalamida, D.; Poulas, K.; Avramopoulou, V.; Fostieri, E.; Lagoumintzis, G.; Lazaridis, K.; Sideri, A.; Zouridakis, M.; Tzartos, S.J. Muscle and neuronal nicotinic acetylcholine receptors. Structure, function and pathogenicity. FEBS J. 2007, 274, 3799–3845. [Google Scholar] [CrossRef] [PubMed]
  5. Rogers, R.S.; Nishimune, H. The role of laminins in the organization and function of neuromuscular junctions. Matrix Biol. 2017, 57–58, 86–105. [Google Scholar] [CrossRef] [PubMed]
  6. Saxena, A.; Stevens, J.; Cetin, H.; Koneczny, I.; Webster, R.; Lazaridis, K. Characterization of an anti-fetal AChR monoclonal antibody isolated from a myasthenia gravis patient. Sci. Rep. 2017, 7, 14426. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  7. Mott, M.; Luna, V.M.; Park, J.Y.; Downes, G.B.; Epley, K.; Ono, F. Expressing acetylcholine receptors after innervation suppresses spontaneous vesicle release and causes muscle fatigue. Sci. Rep. 2017, 7, 1674. [Google Scholar] [CrossRef]
  8. Ching, K.H.; Burbelo, P.D.; Kimball, R.M.; Clawson, L.L.; Corse, A.M.; Iadarola, M.J. Recombinant expression of the nAChR-α1 subunit for the detection of conformation-dependent epitopes in Myasthenia Gravis. Neuromuscul. Disord. 2011, 21, 204–213. [Google Scholar] [CrossRef]
  9. Olivera, B.M.; Teichert, R.W. Diversity of the neurotoxic Conus peptides: A model for concerted pharmacological discovery. Mol. Interv. 2007, 7, 251–260. [Google Scholar] [CrossRef]
  10. Luo, S.; Zhu, X.; Wu, Y.; Zhangsun, D. Conotoxins and Drug Discovery with Special Reference to Hainan Species. In Toxins and Drug Discovery; Springer: Heidelberg, Germany, 2017; pp. 149–187. [Google Scholar]
  11. Azam, L.; McIntosh, J.M. α-Conotoxins as pharmacological probes of nicotinic acetylcholine receptors. Acta Pharmacol. Sin. 2009, 30, 771–783. [Google Scholar] [CrossRef]
  12. Albuquerque, E.X.; Pereira, E.F.; Alkondon, M.; Rogers, S.W. Mammalian nicotinic acetylcholine receptors: From structure to function. Physiol. Rev. 2009, 89, 73–120. [Google Scholar] [CrossRef] [PubMed]
  13. Robinson, S.D.; Norton, R.S. Conotoxin gene superfamilies. Mar. Drugs 2014, 12, 6058–6101. [Google Scholar] [CrossRef] [PubMed]
  14. Dao, F.-Y.; Yang, H.; Su, Z.-D.; Yang, W.; Wu, Y.; Hui, D.; Chen, W.; Tang, H.; Lin, H. Recent Advances in Conotoxin Classification by Using Machine Learning Methods. Molecules 2017, 22, 1057. [Google Scholar] [CrossRef]
  15. Muttenthaler, M.; Akondi, K.B.; Alewood, P.F. Structure-activity studies on α-Conotoxins. Curr. Pharm. Des. 2011, 17, 4226–4241. [Google Scholar] [CrossRef] [PubMed]
  16. Akondi, K.B.; Muttenthaler, M.; Dutertre, S.; Kaas, Q.; Craik, D.J.; Lewis, R.J.; Alewood, P.F. Discovery, synthesis, and structure-activity relationships of Conotoxins. Chem. Rev. 2014, 114, 5815–5847. [Google Scholar] [CrossRef] [PubMed]
  17. Gray, W.R.; Luque, A.; Olivera, B.M. Peptide Toxins from Conus geographus Venom. J. Biol. Chem. 1981, 256, 4734–4740. [Google Scholar] [PubMed]
  18. Johnson, D.S.; Martinez, J.; Eigoyhen, A.B.; Heinemann, S.F.; McIntosh, J.M. α-Conotoxin ImI exhibits subtype-specific nicotinic acetylcholine receptor blockade: Preferential inhibition of homomeric alpha 7 and alpha 9 receptors. Mol. Pharmacol. 1995, 48, 194–199. [Google Scholar]
  19. Pardi, A.; Galdes, A.; Florances, J.; Maniconte, D. Solution Structures of α-Conotoxin GI Determined by Two-Dimensional NMR Spectroscopy. Biochemistry 1989, 28, 5494–5501. [Google Scholar] [CrossRef]
  20. Groebe, D.R.; Dumm, J.M.; Levitan, E.S.; Abramson, S.N. alapha-Conotoxins selectively inhibited one of the two acetylcholine binding sites of nicotinic receptors. Mol. Pharmacol. 1995, 48, 105–111. [Google Scholar]
  21. Hann, R.M.; Pagán, O.R.; Eterović, V.A. The α-Conotoxins GI and MI Distinguish between the Nicotinic Acetylcholine Receptor Agonist Sites while SI Does not. Biochemistry 1994, 33, 14058–14063. [Google Scholar] [CrossRef]
  22. Hann, R.M.; Pagán, O.R.; Gregory, L.M.; Jácome, T.; Eterović, V.A. The 9-arginine residue of alpha-Conotoxin GI is responsible for its selective high affinity for the alphagamma agonist site on the electric organ acetylcholine receptor. Biochemistry 1997, 36, 9051–9056. [Google Scholar] [CrossRef] [PubMed]
  23. Groebe, D.R.; Gray, W.R.; Abramson, S.N. Determinants involved in the affinity of alpha-Conotoxins GI and SI for the muscle subtype of nicotinic acetylcholine receptors. Biochemistry 1997, 36, 6469–6474. [Google Scholar] [CrossRef] [PubMed]
  24. Guddat, L.W.; Martin, J.A.; Shan, L.; Edmundson, A.B.; Gray, W.R. Three-Dimensional Structure of the α-Conotoxin GI at 1.2 Å Resolution. Biochemisty 1996, 35, 11329–11335. [Google Scholar] [CrossRef] [PubMed]
  25. Gehrmann, J.; Alewood, P.F.; Craik, D.J. Structure Determination of the three Disulfide Bond Isomers of α-Conotoxin GI: A Model for the Role of Disulfide Bonds in structural stability. J. Mol. Biol. 1998, 278, 401–415. [Google Scholar] [CrossRef] [PubMed]
  26. Morrison, K.L.; Weiss, G.A. Combinatorial alanine-scanning. Curr. Opin. Chem. Biol. 2001, 5, 302–307. [Google Scholar] [CrossRef]
  27. Eustache, S.; Leprince, J.; Tuffery, P. Progress with peptide scanning to study structure-activity relationships: The implications for drug discovery. Expert Opin. Drugs 2016, 11, 771–784. [Google Scholar] [CrossRef]
  28. Myers, R.A.; Cruz, L.J.; Rivier, J.E.; Olivera, B.M. Conus peptides as chemical probes for receptors and ion channels. Chem. Rev. 1993, 93, 1923–1936. [Google Scholar] [CrossRef]
  29. Myers, R.A.; Zafaralla, G.C.; Gray, W.R.; Abbott, J.; Cruz, L.J.; Olivera, B.M. α-Conotoxins, small peptide probes of nicotinic acetylcholine receptors. Biochemistry 1991, 30, 9370–9377. [Google Scholar] [CrossRef]
  30. Luo, S.; Zhangsun, D.; Zhu, X.; Wu, Y.; Hu, Y.; Christensen, S. Characterization of a novel α-Conotoxin TxID from Conus textile that potently blocks rat alpha3beta4 nicotinic acetylcholine receptors. J. Med. Chem. 2013, 56, 9655–9663. [Google Scholar] [CrossRef]
  31. Loughnan, M.; Nicke, A.; Jones, A.; Schroeder, C.I.; Nevin, S.T.; Adams, D.J. Identification of a novel class of nicotinic receptor antagonists: Dimeric Conotoxins VxXIIA, VxXIIB, and VxXIIC from Conus vexillum. J. Biol. Chem. 2006, 281, 24745–24755. [Google Scholar] [CrossRef]
  32. Luo, S.; Christensen, S.; Zhangsun, D.; Wu, Y.; Hu, Y.; Zhu, X. A Novel Inhibitor of alapha9alapha10 Nicotinic Acetylcholine Receptors from Conus vexillum Delineates a New Conotoxin Superfamily. PLoS ONE 2013, 8, e54648. [Google Scholar]
  33. Luo, S.; Zhangsun, D.; Harvey, P.J.; Kaas, Q.; Wu, Y.; Zhu, X. Cloning, synthesis, and characterization of αO-conotoxin GeXIVA, a potent alapha9alapha10 nicotinic acetylcholine receptor antagonist. Proc. Natl. Acad. Sci. USA 2015, 112, E4026–E4035. [Google Scholar] [CrossRef] [PubMed]
  34. Xu, S.; Zhang, T.; Kompella, S.N.; Yan, M.; Lu, A.; Wang, Y. Conotoxin alphaD-GeXXA utilizes a novel strategy to antagonize nicotinic acetylcholine receptors. Sci. Rep. 2015, 5, 14261. [Google Scholar] [CrossRef] [PubMed]
  35. Jiang, S.; Tae, H.S.; Xu, S.; Shao, X.; Adams, D.J.; Wang, C. Identification of a Novel O-Conotoxin Reveals an Unusual and Potent Inhibitor of the Human alpha9alpha10 Nicotinic Acetylcholine Receptor. Mar. Drugs 2017, 15, 170. [Google Scholar] [CrossRef] [PubMed]
  36. Blount, P.; Merlie, J.P. Molecular Basis of the Two Nonequivalent Ligand Binding Sites of the Muscle Nicotinic Acetylcholine Receptor. Neuron 1989, 3, 349–357. [Google Scholar] [CrossRef]
  37. Kreienkamp, H.J.; Sine, S.M.; Maeda, R.K.; Taylor, P. Glycosylation sites selectively interfere with alpha-toxin binding to the nicotinic acetylcholine receptor. J. Biol. Chem. 1994, 269, 8108–8114. [Google Scholar] [PubMed]
  38. Luo, S.; McIntosh, J.M. Iodo-alpha-Conotoxin MI selectively binds the alpha/delta subunit interface of muscle nicotinic acetylcholine receptors. Biochemistry 2004, 43, 6656–6662. [Google Scholar] [CrossRef] [PubMed]
  39. Bren, N.; Sine, S.M. Hydrophobic Pairwise Interactions Stabilize α-Conotoxin MI in the Muscle Acetylcholine Receptor Binding Site. J. Biol. Chem. 2000, 275, 12692–12700. [Google Scholar] [CrossRef]
  40. Sine, S.M.; Kreienkamp, N.B.; Maeda, R.; Taylort, P. Molecular Dissection of Subunit Interfaces in the Acetylcholine Receptor: Identification of Determinants of α-Conotoxin MI Selectivity. Neuropharmacology 1995, 15, 205–211. [Google Scholar] [CrossRef]
  41. Sugiyama, N.; Marchot, P.; Kawanishi, C.; Osaka, H.; Molles, B.; Sine, S.M. Residues at the Subunit Interfaces of the Nicotinic Acetylcholine Receptor That Contribute to α-Conotoxin MI Binding. Mol. Pharmacol. 1998, 53, 787–794. [Google Scholar] [CrossRef]
  42. Liu, L.; Chew, G.; Hawrot, E.; Chi, C.; Wang, C. Two Potent alapha-3/5 Conotoxins from Piscivorous Conus achatinus. Acta Biol. Biophys. Sin. 2007, 39, 438–444. [Google Scholar] [CrossRef]
  43. Giribaldi, J.; Wilson, D.; Nicke, A.; Laconde, G.; Faucherre, A. Synthesis, Structure and Biological Activity of CIA and CIB, Two α-Conotoxins from the Predation-Evoked Venom of Conus catus. Toxins 2018, 10, 222. [Google Scholar] [CrossRef] [PubMed]
  44. Park, K.H.; Suk, J.E.; Jacobsen, R.; Gray, W.R.; McIntosh, J.M.; Han, K.H. Solution conformation of alpha-Conotoxin EI, a neuromuscular toxin specific for the alpha1/delta subunit interface of torpedo nicotinic acetylcholine receptor. J. Biol. Chem. 2001, 276, 49028–49033. [Google Scholar] [CrossRef] [PubMed]
  45. Lebbe, E.K.; Peigneur, S.; Maiti, M.; Devi, P.; Ravichandran, S.; Lescrinier, E.; Ulens, C.; Waelkens, E.; D’Souza, L.; Herdewijn, P.; et al. Structure-function elucidation of a new α-Conotoxin, Lo1a, from Conus longurionis. J. Biol. Chem. 2014, 289, 9573–9583. [Google Scholar] [CrossRef] [PubMed]
  46. Teichert, R.W.; Rivier, J.; Torres, J.; Dykert, J.; Miller, C.; Olivera, B.M. A uniquely selective inhibitor of the mammalian fetal neuromuscular nicotinic acetylcholine receptor. J. Neurosci. 2005, 25, 732–736. [Google Scholar] [CrossRef] [PubMed]
  47. Liuisma, A.O.; López-Vera, E.; Bulaj, G.; Watkins, M.; Olivera, B.M. Characterization of a novel ψ-Conotoxin from Conus parius. Toxicon 2008, 51, 174–180. [Google Scholar] [CrossRef] [PubMed]
  48. Mitchell, S.S.; Shon, K.J.; Foster, M.P.; Davis, D.R.; Olivera, B.M.; Ireland, C.M. Three-Dimensional Solution Structure of Conotoxin ψ-PIIIE, an Acetylcholine Gated Ion Channel Antagonist. Biochemistry 1998, 37, 1215–1220. [Google Scholar] [CrossRef]
  49. Maslennikov, I.V.; Sobol, A.G.; Gladky, K.V.; Lugovskoy, A.A.; Ostrovsky, A.G.; Tsetlin, V.I.; Ivanov, V.T.; Arseniev, A.S. Two distinct structures of α-Conotoxin GI in aqueous solution. Eur. J. Biochem. 1998, 254, 238–247. [Google Scholar] [CrossRef]
  50. Dutertre, S.; Lewis, R.J. Toxin insights into nicotinic acetylcholine receptors. Biochem. Pharmacol. 2006, 72, 661–670. [Google Scholar] [CrossRef]
  51. Jacobsen, R.B.; DelaCruz, R.G.; Grose, J.H.; McIntosh, J.M.; Yoshikami, D.; Olivera, B.M. Critical Residues Influence the Affinity and Selectivity of α-Conotoxin MI for Nicotinic Acetylcholine Receptors. Biochemisty 1999, 38, 13310–13315. [Google Scholar] [CrossRef]
  52. Benie, A.J.; Whitford, D.; Hargittai, B.; Barany, G.; Janes, R.W. Solution structure of α-Conotoxin SI. FEBS J. 2000, 476, 287–295. [Google Scholar] [CrossRef]
  53. Unwin, N. Refined structure of the nicotinic acetylcholine receptor at 4Å resolution. J. Mol. Biol. 2005, 346, 967–989. [Google Scholar] [CrossRef] [PubMed]
  54. Brejc, K.; Van Dijk, W.J.; Klaassen, R.V.; Schuurmans, M.; Smit, A.B.; Sixma, T.K. Crystal structure of an ACh-binding protein reveals the ligand-binding domain of nicotinic receptors. Nature 2001, 411, 269–276. [Google Scholar] [CrossRef] [PubMed]
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Figure 1. Sequences of α-conotoxin GI and its analogues. Each substituted alanine is labeled in bold and blue. The connectivity of Cysteine (CysI-CysIII, CysII-CysIV) is marked in bold and red. * indicates a C-terminal amide.
Figure 1. Sequences of α-conotoxin GI and its analogues. Each substituted alanine is labeled in bold and blue. The connectivity of Cysteine (CysI-CysIII, CysII-CysIV) is marked in bold and red. * indicates a C-terminal amide.
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Figure 2. The HPLC and ESI-MS profiles of α-conotoxin GI and α-conotoxin [E1A] GI. The peptide GI and [E1A] GI were analyzed on a reverse-phase analytical Vydac C18 column with a solvent gradient from 5% buffer B to 40% buffer B for 20 min where buffer A = 0.075% trifluoroacetic acid (TFA), remainder H2O and buffer B = 0.050% TFA, 90% acetonitrile, remainder H2O. The absorbance was monitored at 214 nm. (A) The HPLC chromatogram of fully oxidized peptide GI; (B) ESI-MS data for GI with an observed monoisotopic mass of 1436.50 Da; (C) the HPLC chromatogram of fully folded peptide [E1A] GI; (D) ESI-MS data for [E1A] GI with an observed monoisotopic mass of 1378.52 Da.
Figure 2. The HPLC and ESI-MS profiles of α-conotoxin GI and α-conotoxin [E1A] GI. The peptide GI and [E1A] GI were analyzed on a reverse-phase analytical Vydac C18 column with a solvent gradient from 5% buffer B to 40% buffer B for 20 min where buffer A = 0.075% trifluoroacetic acid (TFA), remainder H2O and buffer B = 0.050% TFA, 90% acetonitrile, remainder H2O. The absorbance was monitored at 214 nm. (A) The HPLC chromatogram of fully oxidized peptide GI; (B) ESI-MS data for GI with an observed monoisotopic mass of 1436.50 Da; (C) the HPLC chromatogram of fully folded peptide [E1A] GI; (D) ESI-MS data for [E1A] GI with an observed monoisotopic mass of 1378.52 Da.
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Figure 3. The effect on mouse α1β1δε expressed in Xenopus laevis oocytes by GI and alanine-substituted analogues. A bar graph of inhibition of mouse α1β1δε by GI and alanine variants. One-way analysis of variance scatter illustrating the loss or increase in the activity of alanine variants (10 nM) compared to wild peptide using Dunnett’s multiple comparisons test. **** indicates p < 0.0001. All data represent mean ± S.E.M, n = 4–6.
Figure 3. The effect on mouse α1β1δε expressed in Xenopus laevis oocytes by GI and alanine-substituted analogues. A bar graph of inhibition of mouse α1β1δε by GI and alanine variants. One-way analysis of variance scatter illustrating the loss or increase in the activity of alanine variants (10 nM) compared to wild peptide using Dunnett’s multiple comparisons test. **** indicates p < 0.0001. All data represent mean ± S.E.M, n = 4–6.
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Figure 4. Blockade of mouse α1β1δε nAChR by GI, [E1A] GI and [Y11A] GI. Representative current traces showing the inhibition of mouse α1β1δε ACh-evoked currents by GI and [E1A] GI at the concentration of 10 nM (A, B), GI and [Y11A] GI at the concentration of 10 μM (C, D). Xenopus laevis oocytes expressing a given mouse α1β1δε nAChR were at a holding potential of −70 mV and were subjected to a 1 s pulse of ACh every minute as previously described [30]. After control responses to ACh, the oocyte was exposed to toxins for 5 min (arrow). The toxin was then washed out and the response to ACh was again measured. “C” indicates control responses to ACh.
Figure 4. Blockade of mouse α1β1δε nAChR by GI, [E1A] GI and [Y11A] GI. Representative current traces showing the inhibition of mouse α1β1δε ACh-evoked currents by GI and [E1A] GI at the concentration of 10 nM (A, B), GI and [Y11A] GI at the concentration of 10 μM (C, D). Xenopus laevis oocytes expressing a given mouse α1β1δε nAChR were at a holding potential of −70 mV and were subjected to a 1 s pulse of ACh every minute as previously described [30]. After control responses to ACh, the oocyte was exposed to toxins for 5 min (arrow). The toxin was then washed out and the response to ACh was again measured. “C” indicates control responses to ACh.
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Figure 5. The concentration–response curves of mouse α1β1δε nAChR subtype for GI and alanine-substituted analogues. (A) The concentration–response curves for GI and [E1A] GI. peptide [E1A] GI shifted the curves to the left, relative to the parent peptide GI. (B) The concentration–response curves for GI, [P5A] GI, [G8A] GI, [R9A] GI and [Y11A] GI. [P5A] GI, [G8A] GI, [R9A] GI and [Y11A] GI were towards the right compared to the native GI. All data represent mean ± S.E.M, n = 7–10.
Figure 5. The concentration–response curves of mouse α1β1δε nAChR subtype for GI and alanine-substituted analogues. (A) The concentration–response curves for GI and [E1A] GI. peptide [E1A] GI shifted the curves to the left, relative to the parent peptide GI. (B) The concentration–response curves for GI, [P5A] GI, [G8A] GI, [R9A] GI and [Y11A] GI. [P5A] GI, [G8A] GI, [R9A] GI and [Y11A] GI were towards the right compared to the native GI. All data represent mean ± S.E.M, n = 7–10.
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Figure 6. The effect of GI and alanine analogues on different nAChR subtypes expressed in Xenopus laevis oocytes. These mutations were determined at the concentration of 10 μM. A bar graph of the inhibition of nAChR subtypes by GI and its analogues. All data represent mean ± S.E.M, n = 4–6.
Figure 6. The effect of GI and alanine analogues on different nAChR subtypes expressed in Xenopus laevis oocytes. These mutations were determined at the concentration of 10 μM. A bar graph of the inhibition of nAChR subtypes by GI and its analogues. All data represent mean ± S.E.M, n = 4–6.
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Figure 7. α-conotoxin GI and its analogues were tested on neuronal rat α9α10 nAChR subtype expressed in Xenopus laevis oocytes. The representative current traces showing the inhibition of rat α9α10 ACh-evoked currents by GI (A), [E1A] GI (B), [P5A] GI (C) and [G8A] GI (D) respectively. Oocytes were clamped at −70 mV holding potential, and membrane currents were evoked with 10 μM ACh. The inhibition of GI, [E1A] GI and [P5A] GI for rat α9α10 nAChR was 48.4%, 10.5%, 72.3% and 69.8% at the concentration of 10 μM respectively. (E) Concentration–response curves for GI, [P5A] GI and [G8A] GI. All data in (E) represent mean ± S.E.M, n = 5–8.
Figure 7. α-conotoxin GI and its analogues were tested on neuronal rat α9α10 nAChR subtype expressed in Xenopus laevis oocytes. The representative current traces showing the inhibition of rat α9α10 ACh-evoked currents by GI (A), [E1A] GI (B), [P5A] GI (C) and [G8A] GI (D) respectively. Oocytes were clamped at −70 mV holding potential, and membrane currents were evoked with 10 μM ACh. The inhibition of GI, [E1A] GI and [P5A] GI for rat α9α10 nAChR was 48.4%, 10.5%, 72.3% and 69.8% at the concentration of 10 μM respectively. (E) Concentration–response curves for GI, [P5A] GI and [G8A] GI. All data in (E) represent mean ± S.E.M, n = 5–8.
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Figure 8. MD stimulations indicate the binding modes of GI to the interface of αδ subunit respectively. (A) Amino acids around 5 Å radius of the Glu-1 of GI are labeled. (B) Amino acids around 4 Å radius of the Tyr-11 of GI are labeled. The α1(+) subunit is shown in green, δ(−) subunit in cyan and the peptides GI in white.
Figure 8. MD stimulations indicate the binding modes of GI to the interface of αδ subunit respectively. (A) Amino acids around 5 Å radius of the Glu-1 of GI are labeled. (B) Amino acids around 4 Å radius of the Tyr-11 of GI are labeled. The α1(+) subunit is shown in green, δ(−) subunit in cyan and the peptides GI in white.
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Table
Table 1. Recovery time after blockade by α-Conotoxin GI and its analogues at the mouse α1β1δε nAChR.
Table 1. Recovery time after blockade by α-Conotoxin GI and its analogues at the mouse α1β1δε nAChR.
Peptides (10 nM)T50 a (min)Peptides (10 μM)T95 b (min)
GI>7GI>9
[E1A] GI 4[Y11A] GI 2
a Time to 50% current amplitude recovery after toxin washout; GI and [E1A] GI were tested at a concentration of 10 nM. b Time to 95% current amplitude recovery after toxin washout; GI and [E1A] GI were tested at a concentration of 10 μM.
Table
Table 2. IC50 and Hill slope values for inhibiting mouse α1β1δε subtype by α-conotoxin GI and its analogues.
Table 2. IC50 and Hill slope values for inhibiting mouse α1β1δε subtype by α-conotoxin GI and its analogues.
NameIC50 (nM) * α1β1δεnHIC50 Ratio Relative to GI
GI5.86 (5.01–6.86)1.24 (0.93–1.55)1.0
[E1A] GI1.83 (1.55–2.15)1.28 (1.06–1.51)0.3
[N4A] GI4.66 (4.19–5.18)1.49 (1.23–1.76)0.8
[P5A] GI54.72(46.85–63.91)0.84 (0.71–0.97)9.3
[G8A] GI170.60(134.60–216.30)0.94 (0.74–1.13)29.1
[R9A] GI49.79 (45.22–54.81)1.34 (1.13–1.54)8.5
[H10A] GI7.62 (6.78–8.57)1.04 (0.88–1.20)1.3
[Y11A] GI381.20(323.40–449.40)0.90 (0.76–1.05)65.1
[S12A] GI5.39 (4.72–6.15)1.28 (1.01–1.55)0.9
IC50 and nH indicates half inhibitory concentration and Hill Slope respectively. * indicates numbers in parentheses are 95% confidence intervals.
Table
Table 3. IC50 and Hill slope values for inhibiting neuronal rat α9α10 nAChR subtype by α-conotoxin GI and three mutants.
Table 3. IC50 and Hill slope values for inhibiting neuronal rat α9α10 nAChR subtype by α-conotoxin GI and three mutants.
NameIC50 (μM) * rα9α10nHIC50 Ratio Relative to GI
GI9.35 (7.02–12.5)1.35 (0.57–2.15)1
[E1A] GI>10--
[P5A] GI4.14 (3.42–5.03)1.01 (0.79–1.22)0.4
[G8A] GI4.21 (3.21–5.51)1.20 (0.83–1.59)0.5
IC50 and nH indicates half inhibitory concentration and Hill slope respectively. * indicates numbers in parentheses are 95% confidence intervals.
Table
Table 4. The sequences of conotoxins from different species of cone snails blocked muscle-type nAChRs.
Table 4. The sequences of conotoxins from different species of cone snails blocked muscle-type nAChRs.
ConotoxinResourceSequenceTargetReference
α-GIC. geographusECCNPACGRHYSC*muscle[17,18]
α-MIC. magusGRCCHPACGKNYSC*muscle[51]
α-SIC. striatusICCNPACGPKYSC*muscle[52]
α-SIAC. striatusYCCHPACGKNFDC*muscle[20]
α-AC1.1b/CIAC. achatinusNGRCCHPACGKHFSC*muscle, α3β2[42,43]
α-EIC. ermineusRDOCCYHPTCNMSNPQIC*muscle, α3β4, α3β2, α4β2[44]
α-Lo1aC. longurionisEGCCSNPACRTNHPEVCDα7, α3β4, muscle[45]
αA-OIVBC. obscurusCCGVONAACHOCVCKNTC*muscle[46]
αB-VxXXIVAC. vexillumVRCLEKSGAQPNKLFRPPCCQKGα9α10, muscle[31,32]
PSFARHSRCVYYTQSRE*
αD-GeXXAC. generalisDVHRPCQSVRPGRVWGKCCLTα9α10, α7, α3β4, α3β2, [34]
RLCSTMCCARADCTCVYHTWmuscle
RGHGCSCVM*
αO-GeXVIAC. generalisTCRSSGRYCRSPYDRRRRYCRRITDα9α10, α7, α3β4, α3β2, [33]
ACV*muscle
αO-GeXXVIIAC. generalisALMSTGTNYRLLKTCRGSGRYCα9α10, muscle[35]
RSPYDCRRRYCRRISDACV*
Ψ-PrIIIEC. pariusAARCCTYHGSCLKEKmuscle[47,48]
CRRKYCC*
For all conotoxins, an asterisk denotes a carboxyl-terminal carboxamide; O and γ indicates Hydroxyproline and γ-carboxyglutamate (Gla).

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MDPI and ACS Style

Ning, J.; Li, R.; Ren, J.; Zhangsun, D.; Zhu, X.; Wu, Y.; Luo, S. Alanine-Scanning Mutagenesis of α-Conotoxin GI Reveals the Residues Crucial for Activity at the Muscle Acetylcholine Receptor. Mar. Drugs 2018, 16, 507. https://doi.org/10.3390/md16120507

AMA Style

Ning J, Li R, Ren J, Zhangsun D, Zhu X, Wu Y, Luo S. Alanine-Scanning Mutagenesis of α-Conotoxin GI Reveals the Residues Crucial for Activity at the Muscle Acetylcholine Receptor. Marine Drugs. 2018; 16(12):507. https://doi.org/10.3390/md16120507

Chicago/Turabian Style

Ning, Jiong, Rui Li, Jie Ren, Dongting Zhangsun, Xiaopeng Zhu, Yong Wu, and Sulan Luo. 2018. "Alanine-Scanning Mutagenesis of α-Conotoxin GI Reveals the Residues Crucial for Activity at the Muscle Acetylcholine Receptor" Marine Drugs 16, no. 12: 507. https://doi.org/10.3390/md16120507

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