Tae HS et al. (2024), Molecular determinants of the selectivity and p...

XB-ART-61118

J Biol Chem 2024 Jan 26;3011:108017. doi: 10.1016/j.jbc.2024.108017.

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Molecular determinants of the selectivity and potency of α-conotoxin Vc1.1 for human nicotinic acetylcholine receptors.

Tae HS , Hung A , Clark RJ , Adams DJ .

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The α-conotoxins (α-Ctxs) are short, disulfide-rich peptides derived from the venom of the Conus marine snails, primarily acting as antagonists of nicotinic acetylcholine receptors (nAChRs). Specifically, α-Ctx Vc1.1, a 16-amino acid peptide from C. victoriae, competitively antagonizes non-muscle nAChRs, inhibits nicotine-induced currents in bovine chromaffin cells, and alleviates neuropathic pain in rat models. Although Vc1.1 selectively inhibits rat α9α10 nAChRs, its potency and selectivity across human nAChR subtypes remain unresolved. In this study, we assessed the activity of Vc1.1 on human (h) nAChRs heterologously expressed in Xenopus laevis oocytes using the two-electrode voltage clamp technique and simulated interactions using computational modelling. Vc1.1 selectively antagonized homomeric α9 and heteromeric α3β2 nAChRs, with half-maximal inhibitory concentrations (IC50) of 160 nM and 232 nM, respectively. At hα9[N179A]α10, Vc1.1 exhibited a 20-fold decrease in potency compared to hα9α10, due to the loss of hydrogen bonding with Vc1.1-D11. Conversely, Vc1.1 was four-fold more potent at hα3β2[E86A] compared to hα3β2, possibly influenced by the proximal residue β2-K104, as suggested by molecular dynamics (MD) simulations. Additionally, Vc1.1's potency doubled at hα9[N213K]α10, whereas it remained unchanged at hα9[N213R]α10 nAChRs. MD simulations indicate that altered interactions between the mutant hα9 N179A, N213K, or N213R side chains and Vc1.1-D5 may partly explain these changes in potency. The inhibitory action of Vc1.1 at α9-containing nAChRs is particularly relevant given their role in neuroinflammation, presenting a potential therapeutic pathway for alleviating neuropathic and inflammatory pain. This study provides valuable insights for the rational design of Vc1.1-derived α-Ctxs with enhanced nAChR subtype selectivity.

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Genes referenced: ecd vsig1
GO keywords: acetylcholine receptor activity

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	Figure 1. Structure and amino acid sequences of α-conotoxins Vc1.1 and cyclic Vc1.1 (cVc1.1). In these sequences, the cysteine residues are depicted as small spheres, with the sulphur atoms in the disulfide bonds highlighted in yellow. For cVc1.1, a purple linker sequence (GGAAGG) connects the amino and carboxy termini, creating a cyclic form.
	Figure 2. Activity of α-conotoxin Vc1.1 at the human nAChR subtypes. A) Representative ACh (300 μM, 6 μM, and 50 μM, respectively)-evoked currents mediated by hα3β4, hα3β2, and hα9 nAChRs in the absence (black) and presence (red) of 1 μM Vc1.1. B) Bar graph of the inhibition of ACh-evoked peak current amplitude by Vc1.1 (at 1 and 10 μM) across various heterologous human nAChR subtypes (mean ± SD, n = 5-10). The dashed line indicates 50% inhibition of current amplitude. C) Concentration-response relationships of relative ACh-evoked current amplitude mediated by hα3β2, hα3β4, hα6β2β3, hα6β4, and hα9 nAChRs in the presence of α-Ctx Vc1.1 (mean ± SD, n = 5-12). The calculated IC50 and nH values are summarized in Table 1.
	Figure 3. A) Homology model of α-conotoxin Vc1.1 (yellow ribbon) bound to the ECD of hα3β2 (hα3 in white, β2 in green), highlighting key differences with hα3β4. B) Homology model of α-conotoxin Vc1.1 (yellow ribbon) bound to the ECD of hα9, highlighting key differences between the hα9(+) and hα10(+) subunits.
	Figure 4. A) Concentration-response relationships of relative ACh-evoked current amplitude (mean ± SD, n = 3-9) mediated by hα3β2, hα3[E86A]β2, and hα3[K188A]β2 nAChRs were measured across a concentration range of 10 nM to 10 mM ACh. The calculated EC50 and nH values are summarized in Table 2. Docking poses for acetylcholine (cyan and red spheres) and its interactions with α10-F144 and L146 are shown for B) wild-type hα3β2, C) hα3β2[E86A], and D) hα3β2[K188A]. Yellow spiked symbols indicate likely steric hindrance. Predicted binding affinity values are indicated for each. Only the β2(─) subunit is shown for clarity. The quaternary amine of ACh is circled in red.
	Figure 5. A) Concentration-response relationships of relative ACh-evoked current amplitude (mean ± SD, n = 3-8) mediated by hα9α10, hα9[N179A]α10, hα9[N213K]α10, and hα9[N213R]α10 nAChRs were measured across a concentration range of 10 nM to 10 mM ACh. The calculated EC50 and nH values are summarized in Table 2. Docking poses for acetylcholine (cyan and red spheres) and its interactions with α10-R83 and R143 are shown for A) wild-type hα9α10, B) hα9[N213K]α10 (tick symbol indicates favourable contact), and C) hα9[N213R]α10 (yellow spiked symbol indicates likely steric hindrance), with predicted binding affinity values indicated for each. Only the α10(─) subunit is shown for clarity. The quaternary amine of ACh is circled in red.
	Figure 6. A) Concentration–response relationships of relative ACh-evoked current amplitude mediated by hα3β2, hα3β2[E86A], and hα3β2[K188A] nAChRs (mean ± SD, n = 8-12) in the presence of Vc1.1. Whole-cell currents at hα3β2, hα3β2[E86A], and hα3β2[K188A] were activated by 6 μM, 25 μM, and 300 μM ACh, respectively. The calculated IC50 and nH values are summarized in Table 2. B) Time series plots of the percentage change in the number of inter-atomic contacts, relative to the initial homology structure, between Vc1.1 and the β2 (black line) and the β4 (gray line) subunits. C) Number of contacts between β2-K104 (black) or β4-I102 (blue) with Vc1.1-D11.D) Number of contacts between β2-K104 for wt (black), β2[K188A] (blue) and β2[E86A] (red) with Vc1.1-D11.
	Figure 7. A) Concentration–response relationships of relative ACh-evoked current amplitude mediated by hα9α10, hα9[N179A]α10, hα9[N213K]α10, and hα9[N213R]α10 nAChRs (mean ± SD, n = 6-11) in the presence of Vc1.1. Whole-cell currents for hα9α10, hα9[N179A]α10 and hα9[N213R]α10, and hα9[N213K]α10 were activated by 6 μM, 10 μM, and 40 μM ACh, respectively. The calculated IC50 and nH values are summarized in Table 2. B-D) MD simulation structures showing residue 213 of the α9 subunit in contact with Vc1.1 for B) wild-type hα9α10, C) [N213R]-hα9α10, and D) [N213K]-hα9α10. E) Time series plots of the number of inter-atomic contacts between position 213 of α9 and Vc1.1 for wild-type hα9α10 (black), [N213R]-hα9α10 (green), and [N213K]-hα9α10 (red). F-G) MD simulation structures showing residue 179 of the α9 subunit in contact with Vc1.1 for H) wild-type hα9α10 and H) [N213A]-hα9α10. I) Time series plots of the number of inter-atomic contacts between position 179 of α9 and Vc1.1 for wild-type hα9α10 (black) and [N179A]-hα9α10 (blue).
	Figure 1. Structure and amino acid sequences of α-conotoxins Vc1.1 and cyclic Vc1.1 (cVc1.1). In these sequences, the cysteine residues are depicted as small spheres, with the sulphur atoms in the disulfide bonds highlighted in yellow. For cVc1.1, a purple linker sequence (GGAAGG) connects the amino and carboxy termini, creating a cyclic form.
	Figure 2. Activity of α-conotoxin Vc1.1 at the human nAChR subtypes.A, representative ACh (300 μM, 6 μM, and 50 μM, respectively)-evoked currents mediated by hα3β4, hα3β2, and hα9 nAChRs in the absence (black) and presence (red) of 1 μM Vc1.1. B, bar graph of the inhibition of ACh-evoked peak current amplitude by Vc1.1 (at 1 and 10 μM) across various heterologous human nAChR subtypes (mean ± SD, n = 5–10). The dashed line indicates 50% inhibition of current amplitude. C, concentration-response relationships of relative ACh-evoked current amplitude mediated by hα3β2, hα3β4, hα6∗β2β3, hα6∗β4, and hα9 nAChRs in the presence of α-Ctx Vc1.1 (mean ± SD, n = 5–12). The calculated IC50 and nH values are summarized in Table 1.
	Figure 3. Homologymodel of α-conotoxin Vc1.1bound to theextracellular domainof nAChRs.A, ribbon structure of hα3β2 (hα3 in white, β2 in green) bound to Vc1.1 (yellow ribbon), highlighting key differences with hα3β4. B, hα9 bound to Vc1.1, highlighting key differences between the hα9(+) and hα10(+) subunits.
	Figure 4. Concentration-response relationships and molecular models of ACh activation of wild-type and mutant hα3β2 nAChRs.A, Concentration-response relationships of relative ACh-evoked current amplitude (mean ± SD, n = 3–9) mediated by hα3β2, hα 3[E86A]β2, and hα3[K188A]β2 nAChRs were measured across a concentration range of 10 nM to 10 mM ACh. The calculated EC50 and nH values are summarized in Table 2. Docking poses for acetylcholine (cyan and red spheres) and its interactions with α10-F144 and L146 are shown for (B) wild-type hα3β2, (C) hα3β2[E86A], and (D) hα3β2[K188A]. Yellow spiked symbols indicate likely steric hindrance. Predicted binding affinity values are indicated for each. Only the β2(—) subunit is shown for clarity. The quaternary amine of ACh is circled in red.
	Figure 5. Concentration-response relationships and molecular models of ACh activation of wild-type and mutant hα9α10 nAChRs.A, concentration-response relationships of relative ACh-evoked current amplitude (mean ± SD, n = 3–8) mediated by hα9α10, hα9[N179A]α10, hα9[N213K]α10, and hα9[N213R]α10 nAChRs were measured across a concentration range of 10 nM to 10 mM ACh. The calculated EC50 and nH values are summarized in Table 2. Docking poses for acetylcholine (cyan and red spheres) and its interactions with α10-R83 and R143 are shown for (A) wild-type hα9α10, (B) hα9[N213K]α10 (tick symbol indicates favourable contact), and (C) hα9[N213R]α10 (yellow spiked symbol indicates likely steric hindrance), with predicted binding affinity values indicated for each. Only the α10(—) subunit is shown for clarity. The quaternary amine of ACh is circled in red.
	Figure 6. Vc1.1 concentration-response relationship and MD time series for wild-type and mutant hα3-containing nAChRs.A, Concentration–response relationships of relative ACh-evoked current amplitude mediated by hα3β2, hα3β2[E86A], and hα3β2[K188A] nAChRs (mean ± SD, n = 8–12) in the presence of Vc1.1. Whole-cell currents at hα3β2, hα3β2[E86A], and hα3β2[K188A] were activated by 6 μM, 25 μM, and 300 μM ACh, respectively. The calculated IC50 and nH values are summarized in Table 2. B, Time series plots of the percentage change in the number of inter-atomic contacts, relative to the initial homology structure, between Vc1.1 and the β2 (black line) and the β4 (gray line) subunits. C, Number of contacts between β2-K104 (black) or β4-I102 (blue) with Vc1.1-D11. D, Number of contacts between β2-K104 for wt (black), β2[K188A] (blue) and β2[E86A] (red) with Vc1.1-D11.
	Figure 7. Vc1.1 concentration-response relationship and MD data for wild-type and mutant hα9α10 nAChRs.A, Concentration–response relationships of relative ACh-evoked current amplitude mediated by hα9α10, hα9[N179A]α10, hα9[N213K]α10, and hα9[N213R]α10 nAChRs (mean ± SD, n = 6–11) in the presence of Vc1.1. Whole-cell currents for hα9α10, hα9[N179A]α10 and hα9[N213R]α10, and hα9[N213K]α10 were activated by 6 μM, 10 μM, and 40 μM ACh, respectively. The calculated IC50 and nH values are summarized in Table 2. B–D, MD simulation structures showing residue 213 of the α9 subunit in contact with Vc1.1 for (B) wild-type hα9α10, (C) [N213R]-hα9α10, and (D) [N213K]-hα9α10. E, time series plots of the number of inter-atomic contacts between position 213 of α9 and Vc1.1 for wild-type hα9α10 (black), [N213R]-hα9α10 (green), and [N213K]-hα9α10 (red). F and G, MD simulation structures showing residue 179 of the α9 subunit in contact with Vc1.1 for (F) wild-type hα9α10 and (G) [N213A]-hα9α10. H, time series plots of the number of inter-atomic contacts between position 179 of α9 and Vc1.1 for wild-type hα9α10 (black) and [N179A]-hα9α10 (blue).