Ning J et al. (2018), Alanine-Scanning Mutagenesis of α-Conotoxin GI ...

XB-ART-55788

Mar Drugs 2018 Dec 13;1612:. doi: 10.3390/md16120507.

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

Ning J , Li R , Ren J , Zhangsun D , Zhu X , Wu Y , Luo S .

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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, Arg⁸, 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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	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 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 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 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 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 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 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 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.

References [+] :

Akondi, Discovery, synthesis, and structure-activity relationships of conotoxins. 2014, Pubmed