Inhibition of nicotinic acetylcholine receptors, a novel facet in the pleiotropic activities of snake venom phospholipases A2.

Phospholipases A2 represent the most abundant family of snake venom proteins. They manifest an array of biological activities, which is constantly expanding. We have recently shown that a protein bitanarin, isolated from the venom of the puff adder Bitis arietans and possessing high phospholipolytic activity, interacts with different types of nicotinic acetylcholine receptors and with the acetylcholine-binding protein. To check if this property is characteristic to all venom phospholipases A2, we have studied the capability of these enzymes from other snakes to block the responses of Lymnaea stagnalis neurons to acetylcholine or cytisine and to inhibit α-bungarotoxin binding to nicotinic acetylcholine receptors and acetylcholine-binding proteins. Here we present the evidence that phospholipases A2 from venoms of vipers Vipera ursinii and V. nikolskii, cobra Naja kaouthia, and krait Bungarus fasciatus from different snake families suppress the acetylcholine- or cytisine-elicited currents in L. stagnalis neurons and compete with α-bungarotoxin for binding to muscle- and neuronal α7-types of nicotinic acetylcholine receptor, as well as to acetylcholine-binding proteins. As the phospholipase A2 content in venoms is quite high, under some conditions the activity found may contribute to the deleterious venom effects. The results obtained suggest that the ability to interact with nicotinic acetylcholine receptors may be a general property of snake venom phospholipases A2, which add a new target to the numerous activities of these enzymes

Comparison of CM-II and vurtoxin effects on Cyt- and ACh-evoked currents in neurons containing predominantly nAChR-Ls-2 or nAChR-Ls-1.

<p><i>A</i> - A neuron with low sensitivity to ImI against ACh-induced current (nAChR-Ls-2). CM-II at concentration of 500 nM heavily decreased the Cyt-elicited current but affected very slightly the response to ACh. <i>B</i> - Vurtoxin at concentration of 8 µM decreased the currents elicited by Cyt or ACh in two neurons predominantly containing nAChR-Ls2 (up line) or nAChR-Ls1 (middle line) to 0.56 and 0.60 of the controls, respectively. In the third neuron with predominant nAChR-Ls-2 (bottom line) vurtoxin at the same concentration reduced the ACh-evoked current only to 0.80.</p

Affinities for inhibiting different targets and phospholipolytic activities of PLA₂s.

<p>* The data obtained for antagonizing action on currents induced by ACh in nAChR-Ls-1 neurons and by Cyt were combined</p><p>** Not determined</p><p>Affinities for inhibiting different targets and phospholipolytic activities of PLA₂s.</p

Molecular modelling of vurtoxin interaction with <i>Torpedo</i> nAChR.

<p>Vurtoxin active site residues are shown in red. <i>A</i> - Model of vurtoxin spatial structure. C and N indicate C- and N-termini, respectively. <i>B</i> - Model of vurtoxin complex with receptor extracellular domain at the α-γ subunit interface of the <i>T. californica</i> nAChR (vurtoxin is shown in magenta). <i>C</i> - Model of full size <i>Torpedo</i> nAChR complexed with vurtoxin at the α-γ subunit interface.</p

Isolation of vurtoxin and Vur-S49 by reverse-phase HPLC.

<p>Separation was done on a Discovery BIO Wide Pore C18 column (10×250 mm, Supelco) in a gradient of 25–40% (v/v) acetonitrile in 60 min in the presence of 0.1% (v/v) trifluoroacetic acid, at a flow rate of 2.0 ml/min. Fraction containing Vur-S49 (4) and vurtoxin (11) are indicated by horizontal bars.</p

Radioligand analysis.

<p>Inhibition of [¹²⁵I]-labeled α-Bgt binding by CM-II (<i>A</i>), vurtoxin (<i>B</i>) and Vur-PL2 (<i>C</i>) to <i>T. californica</i> (1, crosses, dashed line) and human α7 (3, filled circles, solid line) nAChRs as well as to <i>L. stagnalis</i> (2, open circles, dot line) and <i>A. californica</i> (4, filled squares, dot line) AChBPs. Each point is a mean ± s.e.m value of two or three measurements for each concentration. The curves were calculated from the means ± s.e.m. using ORIGIN 7.5 program. The calculated IC₅₀ values are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115428#pone-0115428-t001" target="_blank">Table 1</a>.</p

Concentration dependences of agonist-evoked currents in <i>L. staganalis</i> neurons.

<p><i>A</i> – Dependence of agonist-evoked currents on concentrations of PLA₂s. 1 (squares) - CM-II from <i>N. kaouthia</i> venom, 2 (triangles) - bitanarin from <i>B. arietans</i>, 3 (filled circles) – vurtoxin, 4 (reversed triangles) - Vur-PL2, 5 (open circles) - Vur-S49 from <i>V. ursinii</i>. IC₅₀ values and Hill coefficients were for CM-II 0.37±0.07 µM and 1.06±0.18, for bitanarin 4.8±1.3 µM and 1.0±0.3, for vurtoxin 10.5±2.6 µM and 3.1±2.7, for Vur-PL2>30 µM, for Vur-S49 2.18±1.28 µM and 1.2±0.89. The data were obtained from 16, 7, 12, 6, and 9 cells, respectively. <i>B</i> –Dependence of the current elicited by either Cyt or ACh on agonist concentrations in control (1, filled circles) and after treatment with 2 µM Vur-S49 (2, open circles). EC₅₀ and Hill slope values of 2.14 µM and 1.83, respectively, in control were changed to 2.38 µM and 2.00 after cell treatment with Vur-S49. The data were obtained from 3 neurons, one containing predominantly nAChR-Ls-1 and two with predominant nAChR-Ls-2, ACh being used on nAChR-Ls-1 neuron and Cyt on two nAChR-Ls-2 cells.</p

Amino acid sequences of 5 from 7 PLA₂s used in the present study.

<p>CM-II (P00596) from <i>N. kaouthia</i>; KBf VI (P00627) from <i>B. fasciatus</i>; vurtoxin (F8QN54), Vur-S49 (F8QN50), and Vur-PL2 (F8QN53) from <i>V. ursinii</i>. Asterisks indicate identical residues.</p

Inhibition of ACh- or Cyt-elicited current in <i>L. stagnalis</i> neurons by PLA₂s from different snakes.

<p><i>A</i> - CM-II from <i>N. kaouthia</i>; <i>B</i> - vurtoxin from <i>V. ursinii</i>; <i>C</i> – KBf VI from <i>B. fasciatus</i>; <i>D</i> – HDP-1 from <i>V. nikolskii</i>. <i>E</i> – Vur-S49 from <i>V. ursinii.</i> Duration of the treatment of the neurons with PLA₂s was 5 min in all cases. The potency of CM-II as the antagonist was approximately the same while used against the ACh- or Cyt-induced current in nAChR-Ls-1 containing neuron (A). Very slow and incomplete recovery of the response to ACh (Cyt) is clearly seen after washing out of CM-II at 2.5 µM (A), vurtoxin at 8 and 25 µM (B) or Vur-S49 at 2 and 10 µM (E).</p