ProTx II

Antihyperalgesic effects of ProTx-II, a Nav1.7 antagonist, and A803467, a Nav1.8 antagonist, in diabetic mice.[Pubmed:27186141]

J Exp Pharmacol. 2015 Jun 24;7:11-6.

The present study investigated the effects of intrathecal administration of ProTx-II (tarantula venom peptide) and A803467 (5-[4-chloro-phenyl]-furan-2-carboxylic acid [3,5-dimethoxy-phenyl]-amide), selective Nav1.7 and Nav1.8 antagonists, respectively, on thermal hyperalgesia in a painful diabetic neuropathy model of mice. Intrathecal administration of ProTx-II at doses from 0.04 to 4 ng to diabetic mice dose-dependently and significantly increased the tail-flick latency. Intrathecal administration of A803467 at doses from 10 to 100 ng to diabetic mice also dose-dependently and significantly increased the tail-flick latency. However, intrathecal administration of either ProTx-II (4 ng) or A803467 (100 ng) had no effect on the tail-flick latency in nondiabetic mice. The expression of either the Nav1.7 or Nav1.8 sodium channel protein in the dorsal root ganglion in diabetic mice was not different from that in nondiabetic mice. The present results suggest that ProTx-II and A803467, highly selective blockers of Nav1.7 and Nav1.8 sodium channels, respectively, in the spinal cord, can have antihyperalgesic effects in diabetic mice.

Interaction of Tarantula Venom Peptide ProTx-II with Lipid Membranes Is a Prerequisite for Its Inhibition of Human Voltage-gated Sodium Channel NaV1.7.[Pubmed:27311819]

J Biol Chem. 2016 Aug 12;291(33):17049-65.

ProTx-II is a disulfide-rich peptide toxin from tarantula venom able to inhibit the human voltage-gated sodium channel 1.7 (hNaV1.7), a channel reported to be involved in nociception, and thus it might have potential as a pain therapeutic. ProTx-II acts by binding to the membrane-embedded voltage sensor domain of hNaV1.7, but the precise peptide channel-binding site and the importance of membrane binding on the inhibitory activity of ProTx-II remain unknown. In this study, we examined the structure and membrane-binding properties of ProTx-II and several analogues using NMR spectroscopy, surface plasmon resonance, fluorescence spectroscopy, and molecular dynamics simulations. Our results show a direct correlation between ProTx-II membrane binding affinity and its potency as an hNaV1.7 channel inhibitor. The data support a model whereby a hydrophobic patch on the ProTx-II surface anchors the molecule at the cell surface in a position that optimizes interaction of the peptide with the binding site on the voltage sensor domain. This is the first study to demonstrate that binding of ProTx-II to the lipid membrane is directly linked to its potency as an hNaV1.7 channel inhibitor.

The tarantula toxins ProTx-II and huwentoxin-IV differentially interact with human Nav1.7 voltage sensors to inhibit channel activation and inactivation.[Pubmed:20855463]

Mol Pharmacol. 2010 Dec;78(6):1124-34.

The voltage-gated sodium channel Na(v)1.7 plays a crucial role in pain, and drugs that inhibit hNa(v)1.7 may have tremendous therapeutic potential. ProTx-II and huwentoxin-IV (HWTX-IV), cystine knot peptides from tarantula venoms, preferentially block hNa(v)1.7. Understanding the interactions of these toxins with sodium channels could aid the development of novel pain therapeutics. Whereas both ProTx-II and HWTX-IV have been proposed to preferentially block hNa(v)1.7 activation by trapping the domain II voltage-sensor in the resting configuration, we show that specific residues in the voltage-sensor paddle of domain II play substantially different roles in determining the affinities of these toxins to hNa(v)1.7. The mutation E818C increases ProTx-II's and HWTX-IV's IC(50) for block of hNa(v)1.7 currents by 4- and 400-fold, respectively. In contrast, the mutation F813G decreases ProTx-II affinity by 9-fold but has no effect on HWTX-IV affinity. It is noteworthy that we also show that ProTx-II, but not HWTX-IV, preferentially interacts with hNa(v)1.7 to impede fast inactivation by trapping the domain IV voltage-sensor in the resting configuration. Mutations E1589Q and T1590K in domain IV each decreased ProTx-II's IC(50) for impairment of fast inactivation by ~6-fold. In contrast mutations D1586A and F1592A in domain-IV increased ProTx-II's IC(50) for impairment of fast inactivation by ~4-fold. Our results show that whereas ProTx-II and HWTX-IV binding determinants on domain-II may overlap, domain II plays a much more crucial role for HWTX-IV, and contrary to what has been proposed to be a guiding principle of sodium channel pharmacology, molecules do not have to exclusively target the domain IV voltage-sensor to influence sodium channel inactivation.

ProTx-II, a selective inhibitor of NaV1.7 sodium channels, blocks action potential propagation in nociceptors.[Pubmed:18728100]

Mol Pharmacol. 2008 Nov;74(5):1476-84.

Voltage-gated sodium (Na(V)1) channels play a critical role in modulating the excitability of sensory neurons, and human genetic evidence points to Na(V)1.7 as an essential contributor to pain signaling. Human loss-of-function mutations in SCN9A, the gene encoding Na(V)1.7, cause channelopathy-associated indifference to pain (CIP), whereas gain-of-function mutations are associated with two inherited painful neuropathies. Although the human genetic data make Na(V)1.7 an attractive target for the development of analgesics, pharmacological proof-of-concept in experimental pain models requires Na(V)1.7-selective channel blockers. Here, we show that the tarantula venom peptide ProTx-II selectively interacts with Na(V)1.7 channels, inhibiting Na(V)1.7 with an IC(50) value of 0.3 nM, compared with IC(50) values of 30 to 150 nM for other heterologously expressed Na(V)1 subtypes. This subtype selectivity was abolished by a point mutation in DIIS3. It is interesting that application of ProTx-II to desheathed cutaneous nerves completely blocked the C-fiber compound action potential at concentrations that had little effect on Abeta-fiber conduction. ProTx-II application had little effect on action potential propagation of the intact nerve, which may explain why ProTx-II was not efficacious in rodent models of acute and inflammatory pain. Mono-iodo-ProTx-II ((125)I-ProTx-II) binds with high affinity (K(d) = 0.3 nM) to recombinant hNa(V)1.7 channels. Binding of (125)I-ProTx-II is insensitive to the presence of other well characterized Na(V)1 channel modulators, suggesting that ProTx-II binds to a novel site, which may be more conducive to conferring subtype selectivity than the site occupied by traditional local anesthetics and anticonvulsants. Thus, the (125)I-ProTx-II binding assay, described here, offers a new tool in the search for novel Na(V)1.7-selective blockers.

Evidence for multiple effects of ProTxII on activation gating in Na(V)1.5.[Pubmed:18657562]

Toxicon. 2008 Sep 1;52(3):489-500.

The peptide toxin ProTxII, recently isolated from the venom of the tarantula spider Thrixopelma pruriens, modifies gating in voltage-gated Na+ and Ca2+ channels. ProTxII is distinct from other known Na+ channel gating modifier toxins in that it affects activation, but not inactivation. It shifts activation gating positively and decreases current magnitude such that the dose-dependence of toxin action measured at a single potential reflects both effects. To test the extent to which these effects were independent, we tracked several different measures of current amplitude, voltage-dependent activation, and current kinetics in Na(V)1.5 in a range of toxin concentrations. Changes in voltage dependence and a decrease in G(max) appeared at relatively low concentrations (40-100 nM) while a positive shift in the voltage range of activation was apparent at higher toxin concentrations (> or =500 nM). Because ProTxII carries a net +4 charge we tested whether electrostatic interactions contributed to toxin action. We examined the effects of ProTxII in the presence of high extracellular Ba2+, known to screen and/or bind to surface charge. Some, but not all aspects of ProTxII modification were sensitive to the presence of Ba2+ indicating the contribution of an electrostatic, surface charge-like mechanism and supporting the idea of a multi-faceted toxin-channel interaction.

Molecular interactions of the gating modifier toxin ProTx-II with NaV 1.5: implied existence of a novel toxin binding site coupled to activation.[Pubmed:17339321]

J Biol Chem. 2007 Apr 27;282(17):12687-97.

Voltage-gated Na(+) channels are critical components in the generation of action potentials in excitable cells, but despite numerous structure-function studies on these proteins, their gating mechanism remains unclear. Peptide toxins often modify channel gating, thereby providing a great deal of information about these channels. ProTx-II is a 30-amino acid peptide toxin from the venom of the tarantula, Thrixopelma pruriens, that conforms to the inhibitory cystine knot motif and which modifies activation kinetics of Na(v) and Ca(v), but not K(v), channels. ProTx-II inhibits current by shifting the voltage dependence of activation to more depolarized potentials and, therefore, differs from the classic site 4 toxins that shift voltage dependence of activation in the opposite direction. Despite this difference in functional effects, ProTx-II has been proposed to bind to neurotoxin site 4 because it modifies activation. Here, we investigate the bioactive surface of ProTx-II by alanine-scanning the toxin and analyzing the interactions of each mutant with the cardiac isoform, Na(v)1.5. The active face of the toxin is largely composed of hydrophobic and cationic residues, joining a growing group of predominantly K(v) channel gating modifier toxins that are thought to interact with the lipid environment. In addition, we performed extensive mutagenesis of Na(v)1.5 to locate the receptor site with which ProTx-II interacts. Our data establish that, contrary to prior assumptions, ProTx-II does not bind to the previously characterized neurotoxin site 4, thus making it a novel probe of activation gating in Na(v) channels with potential to shed new light on this process.

ProTx II is a selective blocker of Nav1.7 sodium channels with an IC50 of 0.3 nM, and is at least 100-fold selective for Nav1.7 over other sodium channel subtypes. ProTx-II inhibits sodium channels by decreasing channel conductance and shifting activation to more positive potentials and blocks action potential propagation in nociceptors.

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