Phlotoxin (PhlTx1, μ-TRTX-Pspp-1) is a neurotoxin from the venom of the tarantula ''

Phlogiellus ''Phlogiellus'' is a genus of tarantulas that was first described by Reginald Innes Pocock in 1897. They are found throughout Asia and Papua New Guinea, including Indonesia, the Philippines, Papua New Guinea, China, Myanmar, Malaysia, Borneo, Thai ...

'' that targets mostly

voltage-sensitive sodium channels Sodium channels are integral membrane proteins that form ion channels, conducting sodium ions (Na+) through a cell's membrane. They belong to the superfamily of cation channels. Classification They are classified into 2 types: Function In exc ...

and mainly

Nav1.7 Sodium voltage-gated channel alpha subunit 9 (also Nav1.7) is a sodium ion channel that, in humans, is encoded by the ''SCN9A'' gene. It is usually expressed at high levels in two types of neurons: the nociceptive (pain) neurons at the dorsal r ...

. The only non-sodium voltage-sensitive channel that is inhibited by Phlotoxin is Kv3.4. Nav1.4 and

Nav1.6 Sodium channel protein type 8 subunit alpha also known as Nav1.6 is a membrane protein encoded by the ''SCN8A'' gene. Nav1.6 is one sodium channel isoform and is the primary voltage-gated sodium channel at each node of Ranvier. The channels are h ...

seem to be Phlotoxin-1-sensitive to some extent as well.

Etymology

Another name for phlotoxin is μ-TRTX-Pspp-1: μ for NaV channel inhibition, then TRTX refers to theraphotoxin which refers to a group of toxins found in the

Theraphosidae Tarantulas comprise a group of large and often hairy spiders of the family Theraphosidae. , 1,100 species have been identified, with 166 genera. The term "tarantula" is usually used to describe members of the family Theraphosidae, although ...

family.

Sources

Phlotoxin was first purified, characterized and sequenced from ''Phlogiellus sp''. ''

'' is a genus of

tarantula Tarantulas comprise a group of large and often hairy spiders of the family Theraphosidae. , 1,100 species have been identified, with 166 genera. The term "tarantula" is usually used to describe members of the family Theraphosidae, although ...

s. Its

venom Venom or zootoxin is a type of toxin produced by an animal that is actively delivered through a wound by means of a bite, sting, or similar action. The toxin is delivered through a specially evolved ''venom apparatus'', such as fangs or a sti ...

, which includes several neurotoxic peptides like phlotoxin, targets diverse

ion channel Ion channels are pore-forming membrane proteins that allow ions to pass through the channel pore. Their functions include establishing a resting membrane potential, shaping action potentials and other electrical signals by Gating (electrophysiol ...

s and chemical receptors.

Chemistry

Structure

Phlotoxin-1 (PhlTx1), weighing around 4058.83 Da, is identified by a 34-

amino acid Amino acids are organic compounds that contain both amino and carboxylic acid functional groups. Although over 500 amino acids exist in nature, by far the most important are the 22 α-amino acids incorporated into proteins. Only these 22 a ...

sequence featuring three

disulfide In chemistry, a disulfide (or disulphide in British English) is a compound containing a functional group or the anion. The linkage is also called an SS-bond or sometimes a disulfide bridge and usually derived from two thiol groups. In inorg ...

bridges and organized based on the Inhibitor Cystine Knot (ICK) architectural motif which is effective for its structural stabilization as three disulfide bonds are structured in a manner where two of them combine to create a circular arrangement, through which the third disulfide bond passes. It is classified to be a member of the NaV channel spider toxin (NaSpTx) family 1. The structure of phlotoxin comprises six

cysteine Cysteine (; symbol Cys or C) is a semiessential proteinogenic amino acid with the chemical formula, formula . The thiol side chain in cysteine enables the formation of Disulfide, disulfide bonds, and often participates in enzymatic reactions as ...

residues forming an ICK architecture fold, with

amidation In organic chemistry, an amide, also known as an organic amide or a carboxamide, is a compound with the general formula , where R, R', and R″ represent any group, typically organyl groups or hydrogen atoms. The amide group is called a p ...

occurring at the

C-terminus The C-terminus (also known as the carboxyl-terminus, carboxy-terminus, C-terminal tail, carboxy tail, C-terminal end, or COOH-terminus) is the end of an amino acid chain (protein Proteins are large biomolecules and macromolecules that comp ...

. "Cys²-Cys¹⁷, Cys⁹-Cys²², Cys¹⁶-Cys²⁹" disulfide bridge organization. The proximity of Cys 16 and 17 makes it challenging to synthesize even though phlotoxin is commercially available.

Homology

The sequence similarity of PhlTx1 with other peptides does not exceed 59%. The closest match regarding inhibition IC₅₀ for PhlTx1 is found in the NaSpTx family to HnTx-III or HwTx-I. It is basically classified under the NaSpTx family, due to the presence of disulfide bridges. PhlTx1 is categorized within the NaSpTx-1 family primarily because of its disulfide bridges. Notably, the inclusion of three

proline Proline (symbol Pro or P) is an organic acid classed as a proteinogenic amino acid (used in the biosynthesis of proteins), although it does not contain the amino group but is rather a secondary amine. The secondary amine nitrogen is in the p ...

residues (Pro11, Pro18, and Pro27) introduces the potential for

Cis–trans isomerism ''Cis''–''trans'' isomerism, also known as geometric isomerism, describes certain arrangements of atoms within molecules. The prefixes "''cis''" and "''trans''" are from Latin: "this side of" and "the other side of", respectively. In the cont ...

. This dynamic property can influence the precise formation of secondary structures and the correct alignment of disulfide bridges, thereby impacting the overall structural integrity of the toxin.

Target

In examining the effects of PhlTx1 on the sodium channel Nav1.7/β1, it appears to share similarities with TTX (tetrodotoxin). Both PhlTx1 and TTX exhibit a capacity to block the channel pore, resulting in a noticeable decrease in sodium currents. Moreover, the behavior of the channel, as reflected in gating parameters, remains largely unaffected by the presence of PhlTx1. This observation suggests a comparable behavior between PhlTx1 and TTX in modulating the function of Nav1.7/β1 channels. The IC₅₀ for PhlTx1 to inhibit Nav1.7 is 39 +/- 2 nM. The PhlTx1 affects all hNav (human voltage-gated Na channels) channels to a different degree except hNav1.8. There is a poor selectivity of PhlTx1 towards the hNav1.1 and 1.3. It also has shown a high affinity towards hNav1.7.

Mode of action

The amino acids which are critical for binding of the hNaV1.7 subtype are identified by their substitution with alanine. When

tryptophan Tryptophan (symbol Trp or W) is an α-amino acid that is used in the biosynthesis of proteins. Tryptophan contains an α-amino group, an α-carboxylic acid group, and a side chain indole, making it a polar molecule with a non-polar aromat ...

at position 24,

lysine Lysine (symbol Lys or K) is an α-amino acid that is a precursor to many proteins. Lysine contains an α-amino group (which is in the protonated form when the lysine is dissolved in water at physiological pH), an α-carboxylic acid group ( ...

at position 25 and

tyrosine -Tyrosine or tyrosine (symbol Tyr or Y) or 4-hydroxyphenylalanine is one of the 20 standard amino acids that are used by cells to synthesize proteins. It is a conditionally essential amino acid with a polar side group. The word "tyrosine" is ...

at position 26 are replaced with alanine, there is a complete loss of affinity. This highlights the critical role of these amino acids in the binding process to Nav1.7. Other substitutions, like alanine at position 1,

serine Serine (symbol Ser or S) is an α-amino acid that is used in the biosynthesis of proteins. It contains an α- amino group (which is in the protonated − form under biological conditions), a carboxyl group (which is in the deprotonated − ...

at position 8, lysine at position 12 or 15, result in a slight change (less than 2.8-fold) in variant affinity, whereas substituting

aspartate Aspartic acid (symbol Asp or D; the ionic form is known as aspartate), is an α-amino acid that is used in the biosynthesis of proteins. The L-isomer of aspartic acid is one of the 22 proteinogenic amino acids, i.e., the building blocks of protein ...

at position 7 leads to an increase in variant affinity (IC₅₀ = 47.0 ± 40.9).

Therapeutic use

Phlotoxin-1 (PhlTx1) has demonstrated selectivity in inhibiting the voltage-gated sodium channel NaV1.7. Its potential as an

antinociceptive In physiology, nociception , also nocioception; ) is the sensory nervous system's process of encoding noxious stimuli. It deals with a series of events and processes required for an organism to receive a painful stimulus, convert it to a molecular ...

agent became apparent when a loss-of-function

mutation In biology, a mutation is an alteration in the nucleic acid sequence of the genome of an organism, virus, or extrachromosomal DNA. Viral genomes contain either DNA or RNA. Mutations result from errors during DNA or viral replication, ...

in the NaV1.7 gene resulted in a congenital inability to perceive pain. Notably, these peptides do not independently exhibit antinociceptive effects; however, when co-administered with exogenous opioids, they bring about analgesic effect, allowing for a significant reduction in opioid dosage. The mechanism underlying the synergistic effect of opioid receptor agonists with selective NaV1.7 inhibitors remains unknown, but this discovery presents a novel approach to pain management. The primary method for evaluating this property involves the formalin test. However, the poor selectivity towards the hNav1.5 and 1.6 subtypes may be associated with in vivo cardiac and neuromuscular side effects, respectively, which could limit its potential use as an analgesic molecule.

References

External links

* * {{cite web , title = Mu-theraphotoxin-Pspp1 , url = https://www.uniprot.org/uniprot/P0DM14 , work = UniProt Peptides Spider toxins Sodium channel blockers Ion channel toxins