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PLoS One
2017 Jan 01;1210:e0186206. doi: 10.1371/journal.pone.0186206.
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Pancreatic and snake venom presynaptically active phospholipases A2 inhibit nicotinic acetylcholine receptors.
Vulfius CA
,
Kasheverov IE
,
Kryukova EV
,
Spirova EN
,
Shelukhina IV
,
Starkov VG
,
Andreeva TV
,
Faure G
,
Zouridakis M
,
Tsetlin VI
,
Utkin YN
.
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Phospholipases A2 (PLA2s) are enzymes found throughout the animal kingdom. They hydrolyze phospholipids in the sn-2 position producing lysophospholipids and unsaturated fatty acids, agents that can damage membranes. PLA2s from snake venoms have numerous toxic effects, not all of which can be explained by phospholipid hydrolysis, and each enzyme has a specific effect. We have earlier demonstrated the capability of several snake venom PLA2s with different enzymatic, cytotoxic, anticoagulant and antiproliferative properties, to decrease acetylcholine-induced currents in Lymnaea stagnalis neurons, and to compete with α-bungarotoxin for binding to nicotinic acetylcholine receptors (nAChRs) and acetylcholine binding protein. Since nAChRs are implicated in postsynaptic and presynaptic activities, in this work we probe those PLA2s known to have strong presynaptic effects, namely β-bungarotoxin from Bungarus multicinctus and crotoxin from Crotalus durissus terrificus. We also wished to explore whether mammalian PLA2s interact with nAChRs, and have examined non-toxic PLA2 from porcine pancreas. It was found that porcine pancreatic PLA2 and presynaptic β-bungarotoxin blocked currents mediated by nAChRs in Lymnaea neurons with IC50s of 2.5 and 4.8 μM, respectively. Crotoxin competed with radioactive α-bungarotoxin for binding to Torpedo and human α7 nAChRs and to the acetylcholine binding protein. Pancreatic PLA2 interacted similarly with these targets; moreover, it inhibited radioactive α-bungarotoxin binding to the water-soluble extracellular domain of human α9 nAChR, and blocked acetylcholine induced currents in human α9α10 nAChRs heterologously expressed in Xenopus oocytes. These and our earlier results show that all snake PLA2s, including presynaptically active crotoxin and β-bungarotoxin, as well as mammalian pancreatic PLA2, interact with nAChRs. The data obtained suggest that this interaction may be a general property of all PLA2s, which should be proved by further experiments.
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29023569
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Fig 1. Inhibition of acetylcholine or cytisine-elicited current in L.stagnalis neurons by PP PLA2 and β-Bgt.(A) Representative recordings from a neuron in control, after a 5-min incubation with PP PLA2 at three different concentrations, and after PP PLA2 wash out. (B) Dependence of ACh- or cytisine-evoked current suppression on PP PLA2 (n = 7) and β-Bgt (n = 4) concentration.
Fig 2. Determination of antagonism type for PP PLA2.Dependence of cytisine (A) or acetylcholine (B) induced currents on agonist concentration in control (closed circles and triangles) and after 5 min treatment with PP PLA2 at 3 μM (open symbols), (n = 5 and 1, respectively).
Fig 3. Inhibition experiments with PP PLA2.(A) Dose-response curve of PP PLA2 inhibitory action on the ACh-evoked (25 μM ACh) ionic currents mediated by human α9α10 nAChR heterologously expressed in Xenopus oocytes. (B) Inhibition by PP PLA2 of the initial rate of specific [125I]-α-Bgt binding to T. californica and human hα7 nAChRs expessed in GH4C1 cells. Only 30% of binding sites in hα7 nAChRs could be protected from [125I]-α-Bgt binding. (C) Inhibition by PP PLA2 of specific [125I]-α-Bgt binding to ECD of human α9 nAChR. IC50 5.5 μM.
Fig 4. Interaction of Cro with nAChR of T. californica electric organ, human neuronal α7 nAChR and AChBP.(A) Inhibition of the initial rate of specific [125I]-α-Bgt binding to T.californica nAChRs by Cro. Points were fit to a 2-site model with affinity for Cro of 30 nM and 260 nM. (B) Inhibition of the initial rate of specific [125I]-α-Bgt binding to human α7 nAChRs by Cro. Two binding sites with affinity for Cro differing more than 3 orders of magnitude were revealed. (C) Inhibition of the initial rate of specific [125I]-α-Bgt binding to acetylcholine-binding protein from L. staganlis by Cro.
Fig 5. SPR recordings of Cro interaction with AChBP from L. stanalis.An arrow indicates injection of the analyte. Line 1 corresponds to injection of buffer solution. Curve 2–5 correspond to injections of solutions with Cro at concentrations of 5.5, 11.5, 23 and 46 μg/ml, respectively.
Fig 6. Interaction of non-venom proteins with T.californica nAChR.Inhibition of the initial rate of specific [125I]-α-Bgt binding to T.californica nAChR by RNAse (squares) and cytochrome C (circles).
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