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Mar Drugs
2018 Nov 22;1612:. doi: 10.3390/md16120460.
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Orthosteric and/or Allosteric Binding of α-Conotoxins to Nicotinic Acetylcholine Receptors and Their Models.
Kryukova EV
,
Ivanov IA
,
Lebedev DS
,
Spirova EN
,
Egorova NS
,
Zouridakis M
,
Kasheverov IE
,
Tzartos SJ
,
Tsetlin VI
.
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α-Conotoxins from Conus snails are capable of distinguishing muscle and neuronal nicotinic acetylcholine receptors (nAChRs). α-Conotoxin RgIA and αO-conotoxin GeXIVA, blocking neuronal α9α10 nAChR, are potential analgesics. Typically, α-conotoxins bind to the orthosteric sites for agonists/competitive antagonists, but αO-conotoxin GeXIVA was proposed to attach allosterically, judging by electrophysiological experiments on α9α10 nAChR. We decided to verify this conclusion by radioligand analysis in competition with α-bungarotoxin (αBgt) on the ligand-binding domain of the nAChR α9 subunit (α9 LBD), where, from the X-ray analysis, αBgt binds at the orthosteric site. A competition with αBgt was registered for GeXIVA and RgIA, IC50 values being in the micromolar range. However, high nonspecific binding of conotoxins (detected with their radioiodinated derivatives) to His₆-resin attaching α9 LBD did not allow us to accurately measure IC50s. However, IC50s were measured for binding to Aplysia californica AChBP: the RgIA globular isomer, known to be active against α9α10 nAChR, was more efficient than the ribbon one, whereas all three GeXIVA isomers had similar potencies at low µM. Thus, radioligand analysis indicated that both conotoxins can attach to the orthosteric sites in these nAChR models, which should be taken into account in the design of analgesics on the basis of these conotoxins.
18-04-00844 Russian Foundation for Basic Research, 17-00-00063 komfi Russian Foundation for Basic Research, 18-04-01366 Russian Foundation for Basic Research
Figure 1. Amino acid sequences of α-conotoxin RgIA and αO-conotoxin GeXIVA with scheme of formation of their three isomers (globular, ribbon and beads) by forming disulfides between 4 cysteine residues (shaded grey and numbered).
Figure 2. HPLC profiles for synthetic peptides used in this study. (A) Co-elution of purified ribbon and globular RgIA isomers in acetonitrile gradient; retention times are shown over respective peaks. (B) Superposition of the RP HPLC profiles of analytical chromatograms for purified globular, beads and ribbon GeXIVA isomers; retention times are shown over respective peaks.
Figure 3. RP HPLC profiles in acetonitrile gradient for the products of the [127I]iodination reaction of α-conotoxin RgIA globular isomer (A) and αO-conotoxin GeXIVA beads isomer (B). The peak of oxidizer (chloramine T, peak 1), non-modified conotoxins (peaks 2), mono- (peaks 3) and di-iodinated derivatives (peak 4) are marked with bars. The numbers above the peaks indicate the corresponding measured molecular masses (Da) obtained by MALDI-TOF mass-spectrometry (A) or electrospray HPLC-MS (B).
Figure 4. Competition of conotoxins (RgIA globular and three GeXIVA isomers) with [125I]-labeled αBgt for binding to α9 LBD. The IC50 values (mean ± SEM) for GeXIVA isomers were 1.140 ± 0.012 μM (globular), 1.20 ± 0.05 μM (ribbon, dotted line) and 1.63 ± 0.02 μM (beads), as well as > 60 μM for RgIA globular isomer.
Figure 5. Inhibition of acetylcholine (10 μM)-evoked currents (A) through rat α9α10 nAChR by globular isomer of RgIA or its [127I]-derivative (5, 100, 300 nM, n = 3) and (B) through human α9α10 nAChR by ribbon isomer GeXIVA or its [127I]-derivative (1, 5, 100, 300 nM, n = 2). The bar graph data are presented as mean ± SEM. (C) and (D) Binding of the radioiodinated conotoxins RgIA globular isomer and GeXIVA beads isomer, respectively, with Ni2+-agarose resin. Binding in the presence (dense pattern bars) or absence (open bars) of α9 ECD is shown. α9 LBD binding with the radioiodinated conotoxins on the Ni2+-NTA-agarose is regarded as 100%. Each bar is the mean ± SEM value of two measurements for each concentration in two (RgIA) or three (GeXIVA) independent experiments.
Figure 6. Inhibition of [125I]-labeled αBgt binding to Aplysia californica AChBP by two RgIA isomers (globular and ribbon) and three GeXIVA isomers (globular, ribbon and beads). The IC50 values were 194 ± 14 nM and 1.00 ± 0.08 μM for the globular and ribbon RgIA analogs, respectively; and around 2.9 μM for all GeXIVA isomers. Each point is the mean ± SEM value of two measurements for each concentration in two independent experiments.
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