Savchenko G. Effect of peptide components of Geolycosa sp venom of the puriergic receptors.

Electrophysiological recordings were carried out on the small DRG neurons demonstrating only fast responses to ATP characteristic of P2X3 receptors. Conventional whole-cell patch-clamp was used in combination with rapid drug application. Using patch clamp and concentration clamp, we have studied the mode of PT1 action on P2X3 receptors in DRG neurons of rat. PT1 produced a small potentiating effect on the current generated by P2X3 receptors. However, when acting on the desensitized receptors, PT1 greatly decreased subsequent responses of P2X3 to ATP. Both effects were totally reversible. We have found that PT1 exerts its inhibitory action by a dramatic concentration-dependent prolongation of P2X3 desensitization removal. In the continuous presence of PT1, inhibition is reversed back to potentiation as the period of agonist applications increases. Desensitized P2X3 receptors acquire a "high-affinity binding site" for the agonist: its unbinding mirrors the rate of recovery from desensitization. Preliminary data suggest that PT1 does not compete for this site acting allosterically. PT1 produced similar effects on human recombinant P2X3 receptors expressed in HEK293 cells. PT1 was ineffective (at concentrations up to 1 µM) against slower responses to ATP generated by the cumulative activity of P2X2 and P2X2/3 receptors. No activity of PT1 against a number of voltage- and ligand-gated channels, including the vanilloid TRPV1 receptors, was found. We have evaluated the effects of PT1 on behavioral reactions of rats in inflammatory pain models. In the Hargreaves plantar test, the antinoceptive effect was observed following intraplantar injection of PT1 into the inflamed hindpaw of rat under both carrageenan- and CFA-induced thermal hyperalgesia. PT1 was also effective in reducing the number of nocifensive events triggered by the injection of capsaicin or formalin. Nocifensive behavior was significantly reduced in the second but not in the first phase of the formalin assay. In summary, as little as only 0.5 nmol of injected peptide produced roughly a two-fold reduction of hyperalgesia in all the employed models. A further four-fold increase in the amount of injected PT1 did not produce additional changes. The analgesic effects of PT1 are in qualitative agreement with observations made using a small molecule, A-317491, which is an antagonist of both P2X3 and P2X2/3 receptors. However, the amount of PT1 per animal used in our experiments is almost 3 orders of magnitude lower than that of A-317491, causing comparable effects. In summary, PT1 is a naturally occurring inhibitor of P2X3 receptors that represents a highly attractive molecular motif for developing new analgesic drugs.