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Front Cell Neurosci
2021 Jan 01;15:765541. doi: 10.3389/fncel.2021.765541.
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Interaction of α9α10 Nicotinic Receptors With Peptides and Proteins From Animal Venoms.
Tsetlin V
,
Haufe Y
,
Safronova V
,
Serov D
,
Shadamarshan P
,
Son L
,
Shelukhina I
,
Kudryavtsev D
,
Kryukova E
,
Kasheverov I
,
Nicke A
,
Utkin Y
.
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Unlike most neuronal nicotinic acetylcholine receptor (nAChR) subunits, α7, α9, and α10 subunits are able to form functional homo- or heteromeric receptors without any β subunits. While the α7 subtype is widely distributed in the mammalian brain and several peripheral tissues, α9 and α9α10 nAChRs are mainly found in the cochlea and immune cells. α-Conotoxins that specifically block the α9α10 receptor showed anti-nociceptive and anti-hyperalgesic effects in animal models. Hence, this subtype is considered a drug target for analgesics. In contrast to the α9α10-selective α-conotoxins, the three-finger toxin α-bungarotoxin inhibits muscle-type and α7 nAChRs in addition to α9α10 nAChRs. However, the selectivity of α-neurotoxins at the α9α10 subtype was less intensively investigated. Here, we compared the potencies of α-conotoxins and α-neurotoxins at the human α9α10 nAChR by two-electrode voltage clamp analysis upon expression in Xenopus oocytes. In addition, we analyzed effects of several α9α10-selective α-conotoxins on mouse granulocytes from bone marrow to identify possible physiological functions of the α9α10 nAChR subtype in these cells. The α-conotoxin-induced IL-10 release was measured upon LPS-stimulation. We found that α-conotoxins RgIA, PeIA, and Vc1.1 enhance the IL-10 expression in granulocytes which might explain the known anti-inflammatory and associated analgesic activities of α9α10-selective α-conotoxins. Furthermore, we show that two long-chain α-neurotoxins from the cobra Naja melanoleuca venom that were earlier shown to bind to muscle-type and α7 nAChRs, also inhibit the α9α10 subtype at nanomolar concentrations with one of them showing a significantly slower dissociation from this receptor than α-bungarotoxin.
FIGURE 1. Influence of nAChR ligands on the release of IL-10 from murine bone marrow granulocytes. Cells were incubated in a medium containing 10 ng/ml lipopolysaccharide from E. coli without or with nicotine or α-conotoxins, as indicated. IL-10 concentrations were measured in supernatants after 23 h of cell incubation using a mouse IL-10 ELISA kit (ab108870, Abcam, United Kingdom). The average values ± SEM of 9–12 independent measurements, each performed in duplicates, are shown. The Kruskal-Wallis One Way Analysis of Variance on Ranks and the Mann-Whitney Rank Sum Test were used. ND, not detectable; *p < 0.05 compared to the cells treated with LPS only.
FIGURE 2. Potencies of snake toxins at the Xenopus laevis oocyte-expressed human α9α10 nicotinic acetylcholine receptor (nAChR). (A) Dose-Response curves and half-maximal inhibitory concentrations (IC50) values of the indicated toxins. Responses to 2 s pulses of 40 μM acetylcholine (ACh) were measured at a potential of –70 mV. Toxins were pre-incubated for 3 min in a static bath. nH: Hill-slope. 95% confidence intervals (CI95) are given in parenthesis. Note that the high values of the Hill coefficients suggest that a 3 min pre-incubation with the toxins is insufficient for complete binding and IC50 values might therefore be underestimated (compare Supplementary Figure 1). However, for practical reasons (decreasing stability of oocytes in the static bath, need of large toxin amounts in case of superfusion), all measurements were performed after 3 min pre-incubation. (B) Recovery of α9α10 current responses after a block induced by 100 nM Tx-NM2 Representative current traces are shown. Black bars indicate application of 40 μM ACh. Interruptions in the traces indicate a 4 min interval. (C) Representative current traces showing the fast dissociation of the indicated toxins from the α9α10 nAChR. Recording conditions are as in (B). Each point represents the mean of 3–5 measurements from different oocytes of least two different frogs. Error bars represent the standard deviation (S.D.).
Figure S1: Influence of antagonist incubation time on Hill slopes of dose inhibition response curves. Human α9α10 nicotinic acetylcholine receptor (nAChR) was expressed in Xenopus laevis oocytes. Oocytes were clamped at -70 mV and activated by 2 s pulses of 40 µM acetylcholine (ACh). Estimated effect of a longer pre-incubation time on the dose inhibition curves of Tx-NM2 and NTI. The solid lines represent dose inhibition curves obtained with a three min pre-incubation time (as presented in Fig. 2A). The dashed lines show a trend towards lower IC50 and Hill slope values with longer (five min) pre-incubation time. Note that only two concentrations per toxin were measured. It can be expected that even longer preincubation times than five min would be required to reach equilibrium binding at low toxin concentrations and the effect of longer preincubation time increases with lower toxin concentrations and would result in a Hill coefficient closer to -1. Mean values with standard deviation are shown. Estimated IC50 values/ Hill slopes for 5 min pre-incubation of Tx-NM2 and NTI are 15 nM/-1.52, and 83 nM/-1.26, respectively. Note that that the estimated IC50 values are in the same order of magnitude for both toxins. n = 3-5 different oocytes for each concentration.
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