Purotoxin-1 (PT-1) is a peptide originally isolated from the Central Asian spider Geolycosa sp. Purotoxin-1 was shown to inhibit selectively P2X3 receptor channels at a 100 nM concentration. Studies were carried-out on cultured rat DRG neurons. Patch-clamp experiments did not show any inhibitory effect of PT-1 on voltage-gated channels (potentials range tested from -100 to 20 mV), neither on TRPV1 (after activation with 500 nM capsaicin). The selectivity of Purotoxin-1 for P2X3 was highlighted by activating this receptor with 10 µM ATP and 100 µM α, β Methylene-ATP. Indeed, unlike P2X3, P2X2 and heterodimer P2X2/3 are known to be not sensitive to such concentrations. Moreover, P2X3, P2X2, and P2X2/3 are the only known ATP-sensitive receptors expressed in plasma membranes of DRG neurons. So, the observed effect seems to be well related to a selective inhibition of P2X3. P2X3-mediated current was fully inhibited with 100 nM Purotoxin-1, making it the most potent and selective ligand for P2X3.

P2X3 receptors are known to be implicated in pain mechanisms. Behavioral experimentations carried-out on rat pain models using 0.5 nmol PT-1 injected intraplantar showed to reduce nociception. This anti-nociceptive effect is comparable to A-317491 compound (Abbott’s drug) with an amount of almost 3 orders of magnitude lower.

Description:

AA sequence: Gly-Tyr-Cys³-Ala-Glu-Lys-Gly-Ile-Arg-Cys¹⁰-Asp-Asp-Ile-His-Cys¹⁵-Cys¹⁶-Thr-Gly-Leu-Lys-Cys²¹-Lys-Cys²³-Asn-Ala-Ser-Gly-Tyr-Asn-Cys³⁰-Val-Cys³²-Arg-Lys-Lys-NH₂Disulfide bridges: Cys³-Cys¹⁶; Cys¹⁰-Cys²¹; Cys¹⁵-Cys³²; Cys²³-Cys³⁰Length (aa): 35Formula: C₁₅₅H₂₄₈N₅₀O₄₈S₈Molecular weight: 3834.59 DaAppearance: White lyophilized solidSolubility: water and saline bufferCAS number:Source: SyntheticPurity rate: > 97 %

Reference:

Modulation of P2X3 receptors by spider toxins

Recently, the novel peptide named purotoxin-1 (PT1) has been identified in the venom of the spider Geolycosa sp. and shown to exert marked modulatory effects on P2X3 receptors in rat sensory neurons. Here we studied another polypeptide from the same spider venom, purotoxin-2 (PT2), and demonstrated that it also affected activity of mammalian P2X3 receptors. The murine and human P2X3 receptors were heterologously expressed in cells of the CHO line, and nucleotide-gated currents were stimulated by CTP and ATP, respectively. Both PT1 and PT2 negligibly affected P2X3-mediated currents elicited by brief pulses of the particular nucleotide. When subthreshold CTP or ATP was added to the bath to exert the high-affinity desensitization of P2X3 receptors, both spider toxins strongly enhanced the desensitizing action of the ambient nucleotides. At the concentration of 50nM, PT1 and PT2 elicited 3-4-fold decrease in the IC(50) dose of ambient CTP or ATP. In contrast, 100nM PT1 and PT2 negligibly affected nucleotide-gated currents mediated by mP2X2 receptors or mP2X2/mP2X3 heteromers. Altogether, our data point out that the PT1 and PT2 toxins specifically target the fast-desensitizing P2X3 receptor, thus representing a unique tool to manipulate its activity.

Purinergic Membrane Receptors as Targets for the Effect of Purotoxin 1, a Component of Venom of Spiders from the Geolycosa Genus

We examined effects of purotoxin 1 (PT1), a component of the venom of Geolycosa spiders, on a few voltageand ligand-operated ion channels present in the plasma membrane of sensory neurons from the rat dorsal root ganglia (DRGs). Purotoxin 1 in a 100 nM concentration evoked no changes in ion currents through voltage-operated sodium, potassium, and calcium channels in the membranes of isolated sensory neurons. This agent was also found to be ineffective with respect to capsaicin-sensitive receptor-channel complexes (TRPV1). Testing of the effects of PT1 on purinergic receptor-channel complexes P2X3, P2X2, and P2X2/3 showed that this toxin is a highly selective blocker of exclusively P2X3 receptors. The selectivity of action of PT1 demonstrated in our experiments shows that it is a unique agent, which opens up new prospects in the studies of structural/functional peculiarities of receptor-channel complexes P2X3 as a peripheral link of the nociception system.

G. A. Savchenko, et al. (2010) Purinergic Membrane Receptors as Targets for the Effect of Purotoxin 1, a Component of Venom of Spiders from the Geolycosa Genus. Neurophysiology.

Novel peptide from spider venom inhibits P2X3 receptors and inflammatory pain

P2X3 purinoreceptors expressed in mammalian sensory neurons play a key role in several processes, including pain perception. From the venom of the Central Asian spider Geolycosa sp., we have isolated a novel peptide, named purotoxin-1 (PT1), which is to our knowledge the first natural molecule exerting powerful and selective inhibitory action on P2X3 receptors. PT1 dramatically slows down the removal of desensitization of these receptors. The peptide demonstrates potent antinociceptive properties in animal models of inflammatory pain.

E.V. Grishin, et al. (2010) Novel peptide from spider venom inhibits P2X3 receptors and inflammatory pain. ANN NEUROL. PMID: 20437566