Protoxin-I

Protoxin-I, also known as ProTx-I, or Beta/omega-theraphotoxin-Tp1a, is a 35-amino-acid peptide neurotoxin extracted from the venom of the tarantula Thrixopelma pruriens. Protoxin-I belongs to the inhibitory cystine knot (ICK) family of peptide toxins, which have been known to potently inhibit voltage-gated ion channels. Protoxin-I selectively blocks low voltage threshold T-type calcium channels,[1][2] voltage gated sodium channels[3][4] and the nociceptor cation channel TRPA1.[5] Due to its unique ability to bind to TRPA1, Protoxin-I has been implicated as a valuable pharmacological reagent with potential applications in clinical contexts with regards to pain and inflammation[5]

Origin

Protoxin-I is a toxin extracted from the venom of the tarantula spider Thrixopelma pruriens, also known as the Peruvian green velvet spider. It is used by the spider to immobilise and catch prey.[6] The primary structure of the mature toxin peptide is homologous to that of Beta/omega-theraphotoxin-Bp1a from the tarantula spider Bumba pulcherrimaklaasi, suggesting a common toxin within the subfamily Theraphosinae.[7]

Structure and active site

Protoxin-I is a peptide possessing a 35 amino acid sequence of Glu-Cys-Arg-Tyr-Trp-Leu-Gly-Gly-Cys-Ser-Ala-Gly-Gln-Thr-Cys-Cys-Lys-His-Leu-Val-Cys-Ser-Arg-Arg-His-Gly-Trp-Cys-Val-Trp-Asp-Gly-Thr-Phe-Ser (see Table 1 for one-letter sequence) with 3 disulphide bonds between Cys2-Cys16, Cys9-Cys21 and Cys15-Cys28.[7] Nuclear Magnetic Resonance Spectroscopy of Protoxin-I revealed two beta strands in the protein structure.[5] Gating-modifier toxins isolated from spider venom all share a conserved molecular structure consisting of a hydrophobic patch, populated by hydrophobic residues, and surrounded by a ring of positively- and negatively charged residues that promote the binding of the peptide to the lipid membrane.[8]

Table 1: Protoxin-I Sequence
1 5 10 15 20 25 30 35
E C R Y W L G G C S A G Q T C C K H L V C S R R H G W C V W D G T F S
Surface profile of Protoxin-I coloured in accordance to residue characteristic: hydrophobic (green), positively charged (blue), negatively charged (red), uncharged (grey).

By systematically exchanging single amino acids by an alanine, features responsible for Protoxin-I toxicity can be revealed. As such, the replacements R3A, W5A, K17E, L19A, S22A, R23A, W27A, V29A, W30A, D31A, G31A, F34A reduce the toxin’s ability to inhibit sodium channels NaV1.2[5][7].  Similarly, the replacements L6A, L19A, W27A, V29A, W30A, D31A also reduce its inhibitory effects on NaV1.7 sodium channels.[7] Lastly, the replacements Q13A, L19A, S22A, W30A, F34A, S35A reveal the active sites for Protoxin-I binding to TRPA1 receptors.[5] These loss-of-function replacements primarily represent residues in the hydrophobic patch and positively- and negatively charged rings, further supporting the idea that these regions play an important role in ion channel binding.[8]

Toxicodynamics

Like other gating-modifier spider toxins, Protoxin-I preferentially binds to anionic lipid-containing membranes where it exhibits complex allosteric interactions with ion channel voltage sensor domains.[9][10] After binding to the lipid membrane, Protoxin-I adopts a shallow position on the anionic membrane with the help of tryptophan residues within the hydrophobic patch,[8] where the positively- and negatively charged rings on the toxin are then able to bind to conserved sequences of hydrophobic and anionic residues on voltage gated ion channels.[11][12]

Voltage-gated sodium channels

Similar to other gating-modifier toxins of the ICK family, Protoxin-I works by shifting the voltage dependence of activation of voltage-gated sodium channels to more positive potentials.[3] Protoxin-I differs from other ICK toxins as Protoxin-I exhibits little selectivity between sodium channels, and is thus able to potently inhibit TTX-resistant sodium currents in sensory neurons through interaction with the Nav1.8 channel.[3][4] Protoxin-I has been found to potently bind to NaV1.2, NaV1.6, NaV1.7 and NaV1.8.[3][7] The pore-forming α subunit of voltage-gated sodium channels consists of 4 homologous domains, each having their own voltage sensor domain. Research has shown that Protoxin-I interacts with the voltage sensor domains of domain II and domain IV, thereby functionally inhibiting the channel.[7] Protoxin-I promiscuity in binding to voltage-gated sodium channels is believed to be because of the high amount of positively charged residues on the peptide surface, which bind to conserved amino acid sequences on the surface of the voltage-sensor domain on sodium channels.[8] This notion is further back by the fact that Protoxin-I binds less potently to NaV1.4 and NaV1.5, which exhibit relatively fewer anionic residues on the voltage sensor domain.[8]

Voltage-Gated T-type calcium channels

Protoxin-I has also been found to shift the voltage dependence of activation of the T-type calcium channels.[1][3] In particular, Protoxin-I is able to differentiate CaV3.1 channels from other human T-type calcium channels, exhibiting a 160-fold increase in potency over that of CaV3.2,[1] and a 10-fold increase in potency over that of CaV3.3.[2] Protoxin-I shifts the voltage dependence of activation of T-type calcium channels to more positive potentials, without changing its voltage dependence of inactivation.[3] Through the use of chimeric channel proteins, the S3-S4 linker on domain IV of the CaV3.1 channel protein has been identified in having greater sensitivity towards Protoxin-I, suggesting that this domain exhibits specific residues that are susceptible to Protoxin-I binding.[1][2]

Transient receptor potential ankyrin 1 (TRPA1)

Protoxin was identified to be the first high-affinity peptide TRPA1 antagonist.[5] TRPA1 is a primary nociceptor channel expressed on the plasma membrane of many human and animal cells. NaV1.2 and TRPA1 were found to have partially homologous binding sites by which Protoxin-I binds to these ion channels. These binding surfaces are, similar to that of sodium channels, located on the extracellular loops of the S1-S4 gating domain of the TRPA1 channel.[5]

Therapeutic uses

In vivo testing in mice revealed that intrathecal injection of Protoxin-I reduces the response to formalin in acute pain and inflammation without signs of neurotoxicity.[13]

Modifications to Protoxin-I can induce altered specificity in binding, producing peptides that only bind to either the TRPA1 or NaV1.2 gating mechanism.[5] This may provide a deeper insight into the biophysiological function and mechanisms underlying TRPA1, with potential clinical applications in pain and inflammation treatment. TRPA1 represents the final common pathway for many pronociceptive induced pathophysiological pain, therefore, pain therapy using TRPA1 antagonists can be expected to have applicable pharmacological use[14]

References
  1. Ohkubo, Tsuyako; Yamazaki, Jun; Kitamura, Kenji (2010). "Tarantula Toxin ProTx-I Differentiates Between Human T-type Voltage-Gated Ca2+ Channels Cav3.1 and Cav3.2". Journal of Pharmacological Sciences. 112 (4): 452–458. doi:10.1254/jphs.09356FP. PMID 20351484. S2CID 29721634.
  2. Bladen, Chris; Hamid, Jawed; Souza, Ivana A.; Zamponi, Gerald W. (2014-05-09). "Block of T-type calcium channels by protoxins I and II". Molecular Brain. 7 (1): 36. doi:10.1186/1756-6606-7-36. ISSN 1756-6606. PMC 4030284. PMID 24886690.
  3. Middleton, Richard E.; Warren, Vivien A.; Kraus, Richard L.; Hwang, Jeremy C.; Liu, Chou J.; Dai, Ge; Brochu, Richard M.; Kohler, Martin G.; Gao, Ying-Duo; Garsky, Victor M.; Bogusky, Michael J. (2002-12-01). "Two Tarantula Peptides Inhibit Activation of Multiple Sodium Channels". Biochemistry. 41 (50): 14734–14747. doi:10.1021/bi026546a. ISSN 0006-2960. PMID 12475222.
  4. Priest, Birgit T.; Blumenthal, Kenneth M.; Smith, Jaime J.; Warren, Vivien A.; Smith, McHardy M. (2007-02-01). "ProTx-I and ProTx-II: Gating modifiers of voltage-gated sodium channels". Toxicon. Gating Modifier Peptides of Ion Channels. 49 (2): 194–201. doi:10.1016/j.toxicon.2006.09.014. ISSN 0041-0101. PMID 17087985.
  5. Gui, Junhong; Liu, Boyi; Cao, Guan; Lipchik, Andrew M.; Perez, Minervo; Dekan, Zoltan; Mobli, Mehdi; Daly, Norelle L.; Alewood, Paul F.; Parker, Laurie L.; King, Glenn F. (2014-03-03). "A Tarantula-Venom Peptide Antagonizes the TRPA1 Nociceptor Ion Channel by Binding to the S1–S4 Gating Domain". Current Biology. 24 (5): 473–483. doi:10.1016/j.cub.2014.01.013. ISSN 0960-9822. PMC 3949122. PMID 24530065. S2CID 7479469.
  6. King, Glenn F.; Hardy, Margaret C. (2013-01-07). "Spider-Venom Peptides: Structure, Pharmacology, and Potential for Control of Insect Pests". Annual Review of Entomology. 58 (1): 475–496. doi:10.1146/annurev-ento-120811-153650. ISSN 0066-4170. PMID 23020618.
  7. Rupasinghe, Darshani B.; Herzig, Volker; Vetter, Irina; Dekan, Zoltan; Gilchrist, John; Bosmans, Frank; Alewood, Paul F.; Lewis, Richard J.; King, Glenn F. (2020-11-01). "Mutational analysis of ProTx-I and the novel venom peptide Pe1b provide insight into residues responsible for selective inhibition of the analgesic drug target NaV1.7". Biochemical Pharmacology. Pharmacology and Therapeutic Potential of Venom Peptides. 181: 114080. doi:10.1016/j.bcp.2020.114080. ISSN 0006-2952. PMID 32511987. S2CID 219549375.
  8. Agwa, Akello J.; Peigneur, Steve; Chow, Chun Yuen; Lawrence, Nicole; Craik, David J.; Tytgat, Jan; King, Glenn F.; Henriques, Sónia Troeira; Schroeder, Christina I. (June 2018). "Gating modifier toxins isolated from spider venom: Modulation of voltage-gated sodium channels and the role of lipid membranes". Journal of Biological Chemistry. 293 (23): 9041–9052. doi:10.1074/jbc.RA118.002553. PMC 5995520. PMID 29703751.
  9. Shen, Huaizong; Li, Zhangqiang; Jiang, Yan; Pan, Xiaojing; Wu, Jianping; Cristofori-Armstrong, Ben; Smith, Jennifer J.; Chin, Yanni K. Y.; Lei, Jianlin; Zhou, Qiang; King, Glenn F. (2018-10-19). "Structural basis for the modulation of voltage-gated sodium channels by animal toxins". Science. 362 (6412): eaau2596. doi:10.1126/science.aau2596. ISSN 0036-8075. PMID 30049784. S2CID 206667555.
  10. Deplazes, Evelyne; Henriques, Sónia Troeira; Smith, Jennifer J.; King, Glenn F.; Craik, David J.; Mark, Alan E.; Schroeder, Christina I. (2016-04-01). "Membrane-binding properties of gating modifier and pore-blocking toxins: Membrane interaction is not a prerequisite for modification of channel gating". Biochimica et Biophysica Acta (BBA) - Biomembranes. 1858 (4): 872–882. doi:10.1016/j.bbamem.2016.02.002. ISSN 0005-2736. PMID 26850736.
  11. Minassian, Natali A.; Gibbs, Alan; Shih, Amy Y.; Liu, Yi; Neff, Robert A.; Sutton, Steven W.; Mirzadegan, Tara; Connor, Judith; Fellows, Ross; Husovsky, Matthew; Nelson, Serena (August 2013). "Analysis of the Structural and Molecular Basis of Voltage-sensitive Sodium Channel Inhibition by the Spider Toxin Huwentoxin-IV (μ-TRTX-Hh2a)". Journal of Biological Chemistry. 288 (31): 22707–22720. doi:10.1074/jbc.M113.461392. PMC 3829356. PMID 23760503.
  12. Cai, Tianfu; Luo, Ji; Meng, Er; Ding, Jiuping; Liang, Songping; Wang, Sheng; Liu, Zhonghua (June 2015). "Mapping the interaction site for the tarantula toxin hainantoxin-IV (β-TRTX-Hn2a) in the voltage sensor module of domain II of voltage-gated sodium channels". Peptides. 68: 148–156. doi:10.1016/j.peptides.2014.09.005. PMID 25218973. S2CID 5610614.
  13. Nicolas, Sébastien; Zoukimian, Claude; Bosmans, Frank; Montnach, Jérôme; Diochot, Sylvie; Cuypers, Eva; De Waard, Stephan; Béroud, Rémy; Mebs, Dietrich; Craik, David; Boturyn, Didier (2019-06-21). "Chemical Synthesis, Proper Folding, Nav Channel Selectivity Profile and Analgesic Properties of the Spider Peptide Phlotoxin 1". Toxins. 11 (6): 367. doi:10.3390/toxins11060367. ISSN 2072-6651. PMC 6628435. PMID 31234412.
  14. Koivisto, Ari; Jalava, Niina; Bratty, Raymond; Pertovaara, Antti (2018-11-01). "TRPA1 Antagonists for Pain Relief". Pharmaceuticals. 11 (4): 117. doi:10.3390/ph11040117. ISSN 1424-8247. PMC 6316422. PMID 30388732.
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