Protoxin I (ProTx-I; β-theraphotoxin-Tp1a) is a toxin that was originally isolated from the venom of Thrixopelma pruriens (Peruvian green velvet tarantula). This toxin reversibly inhibits the tetrodotoxin (TTX)-resistant channel Na_v1.8(IC₅₀ = 27 nM) and Na_v1.2, Na_v1.5 and Na_v1.7 with IC₅₀ values between 50 and 100 nM. Furthermore, ProTx-I shifts the voltage dependence activity of T-type Ca_v3.1 channels (IC₅₀= 50 nM) without affecting the voltage dependence of inactivation. ProTx-I is a valuable tool to discriminate between Ca_v3.1 and Ca_v3.2

Description:

AA sequence: Glu-Cys²-Arg-Tyr-Trp-Leu-Gly-Gly-Cys⁹-Ser-Ala-Gly-Gln-Thr-Cys¹⁵-Cys¹⁶-Lys-His-Leu-Val-Cys²¹-Ser-Arg-Arg-His-Gly-Trp-Cys²⁸-Val-Trp-Asp-Gly-Thr-Phe-Ser-OHDisulfide bridges: Cys²-Cys¹⁶, Cys⁹-Cys²¹, Cys¹⁵-Cys²⁸Length (aa): 35Formula: C₁₇₁H₂₄₅N₅₃O₄₇S₆Molecular Weight: 3987.50 DaAppearance: White lyophilized solidSolubility: water or saline bufferCAS number: Not availableSource: SyntheticPurity rate: > 95 %

Reference:

Two tarantula peptides inhibit activation of multiple sodium channels

Two peptides, ProTx-I and ProTx-II, from the venom of the tarantula Thrixopelma pruriens, have been isolated and characterized. These peptides were purified on the basis of their ability to reversibly inhibit the tetrodotoxin-resistant Na channel, Na(V) 1.8, and are shown to belong to the inhibitory cystine knot (ICK) family of peptide toxins interacting with voltage-gated ion channels. The family has several hallmarks: cystine bridge connectivity, mechanism of channel inhibition, and promiscuity across channels within and across channel families. The cystine bridge connectivity of ProTx-II is very similar to that of other members of this family, i.e., C(2) to C(16), C(9) to C(21), and C(15) to C(25). These peptides are the first high-affinity ligands for tetrodotoxin-resistant peripheral nerve Na(V) channels, but also inhibit other Na(V) channels (IC(50)’s < 100 nM). ProTx-I and ProTx-II shift the voltage dependence of activation of Na(V) 1.5 to more positive voltages, similar to other gating-modifier ICK family members. ProTx-I also shifts the voltage dependence of activation of Ca(V) 3.1 (alpha(1G), T-type, IC(50) = 50 nM) without affecting the voltage dependence of inactivation. To enable further structural and functional studies, synthetic ProTx-II was made; it adopts the same structure and has the same functional properties as the native peptide. Synthetic ProTx-I was also made and exhibits the same potency as the native peptide. Synthetic ProTx-I, but not ProTx-II, also inhibits K(V) 2.1 channels with 10-fold less potency than its potency on Na(V) channels. These peptides represent novel tools for exploring the gating mechanisms of several Na(V) and Ca(V) channels.

Middelton R.E, et al. (2002) Two tarantula peptides inhibit activation of multiple sodium channels. Biochemestry. PMID: 12475222

ProTx-I and ProTx-II: gating modifiers of voltage-gated sodium channels

The tarantula venom peptides ProTx-I and ProTx-II inhibit voltage-gated sodium channels by shifting their voltage dependence of activation to a more positive potential, thus acting by a mechanism similar to that of potassium channel gating modifiers such as hanatoxin and VSTX1. ProTx-I and ProTx-II inhibit all sodium channel (Nav1) subtypes tested with similar potency and represent the first potent peptidyl inhibitors of TTX-resistant sodium channels. Like gating modifiers of potassium channels, ProTx-I and ProTx-II conform to the inhibitory cystine knot motif, and ProTx-II was demonstrated to bind to sodium channels in the closed state. Both toxins have been synthesized chemically, and ProTx-II, produced by recombinant means, has been used to map the interaction surface of the peptide with the Nav1.5 channel. In comparison, beta-scorpion toxins activate sodium channels by shifting the voltage dependence of activation to more negative potentials, and together these peptides represent valuable tools for exploring the gating mechanism of sodium channels.

Priest B.T., et al. (2007) ProTx-I and ProTx-II: gating modifiers of voltage-gated sodium channels. Toxicon. PMID: 17087985

Tarantula toxin ProTx-I differentiates between human T-type voltage-gated Ca²⁺ Channels Ca_v3.1 and Ca_v3.2

ProTx-I peptide, a venom toxin of the tarantula Thrixopelma pruriens, has been reported to interact with voltage-gated ion channels. ProTx-I reduced Ba(2+) currents through recombinant human T-type voltage-gated Ca(2+) channels, Ca(v)3.1 (hCa(v)3.1), with roughly 160-fold more potency than through hCa(v)3.2 channels. Chimeric channel proteins (hCa(v)3.1/S3S4 and hCa(v)3.2/S3S4) were produced by exchanging fourteen amino acids in the hCa(v)3.1 domain IV S3-S4 linker region and the corresponding region of hCa(v)3.2 between each other. The ProTx-I sensitivity was markedly reduced in the hCa(v)3.1/S3S4 chimera as compared to the original hCa(v)3.1 channel, while the hCa(v)3.2/S3S4 chimera exhibited greater ProTx-I sensitivity than the original hCa(v)3.2 channel. These results suggest that the domain IV S3-S4 linker in the hCa(v)3.1 channel may contain residues involved in the interaction of ProTx-I with T-type Ca(2+) channels.

Ohkubo T, et al. (2010) Tarantula toxin ProTx-I differentiates between human T-type voltage-gated Ca²⁺ Channels Ca_v3.1 and Ca_v3.2. J Pharmacol Sci. PMID: 20351484

A Tarantula-Venom Peptide Antagonizes the TRPA1 Nociceptor Ion Channel by Binding to the S1-S4 Gating Domain

BACKGROUND:

The venoms of predators have been an excellent source of diverse highly specific peptides targeting ion channels. Here we describe the first known peptide antagonist of the nociceptor ion channel transient receptor potential ankyrin 1 (TRPA1).

RESULTS:

We constructed a recombinant cDNA library encoding ∼100 diverse GPI-anchored peptide toxins (t-toxins) derived from spider venoms and screened this library by coexpression in Xenopus oocytes with TRPA1. This screen resulted in identification of protoxin-I (ProTx-I), a 35-residue peptide from the venom of the Peruvian green-velvet tarantula, Thrixopelma pruriens, as the first known high-affinity peptide TRPA1 antagonist. ProTx-I was previously identified as an antagonist of voltage-gated sodium (NaV) channels. We constructed a t-toxin library of ProTx-I alanine-scanning mutants and screened this library against NaV1.2 and TRPA1. This revealed distinct partially overlapping surfaces of ProTx-I by which it binds to these two ion channels. Importantly, this mutagenesis yielded two novel ProTx-I variants that are only active against either TRPA1or NaV1.2. By testing its activity against chimeric channels, we identified the extracellular loops of the TRPA1 S1-S4 gating domain as the ProTx-I binding site.

CONCLUSIONS:

These studies establish our approach, which we term “toxineering,” as a generally applicable method for isolation of novel ion channel modifiers and design of ion channel modifiers with altered specificity. They also suggest that ProTx-I will be a valuable pharmacological reagent for addressing biophysical mechanisms of TRPA1 gating and the physiology of TRPA1 function in nociceptors, as well as for potential clinical application in the context of pain and inflammation.

Gui J, et al. (2014) A Tarantula-Venom Peptide Antagonizes the TRPA1 Nociceptor Ion Channel by Binding to the S1-S4 Gating Domain. Curr Biol. PMID: 24530065