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Voltage-gated sodium channels (VGSCs, or Nav) are important determinants of action potential generation and propagation. Efforts are underway to develop medicines targeting different channel subtypes for the treatment of related channelopathies. However, a high degree of conservation across its nine subtypes could lead to the off-target adverse effects on skeletal and cardiac muscles due to acting on primary skeletal muscle sodium channel Nav1.4 and cardiac muscle sodium channel Nav1.5, respectively. For a long evolutionary process, some peptide toxins from venoms have been found to be highly potent yet selective on ion channel subtypes and, therefore, hold the promising potential to be developed into therapeutic agents. In this research, all-atom molecular dynamic methods were used to elucidate the selective mechanisms of an analgesic-antitumor β-scorpion toxin (AGAP) with human Nav1.4 and Nav1.5 in order to unravel the primary reason for the production of its adverse reactions on the skeletal and cardiac muscles. Our results suggest that the rational distribution of residues with ring structures near position 38 and positive residues in the C-terminal on AGAP are critical factors to ensure its analgesic efficacy. Moreover, the substitution for residues with benzene is beneficial to reduce its side effects. [ABSTRACT FROM AUTHOR]

Voltage-gated sodium channels (VGSCs, or Nav) are important determinants of action potential generation and propagation. Efforts are underway to develop medicines targeting different channel subtypes for the treatment of related channelopathies. However, a high degree of conservation across its nine subtypes could lead to the off-target adverse effects on skeletal and cardiac muscles due to acting on primary skeletal muscle sodium channel Nav1.4 and cardiac muscle sodium channel Nav1.5, respectively. For a long evolutionary process, some peptide toxins from venoms have been found to be highly potent yet selective on ion channel subtypes and, therefore, hold the promising potential to be developed into therapeutic agents. In this research, all-atom molecular dynamic methods were used to elucidate the selective mechanisms of an analgesic-antitumor β-scorpion toxin (AGAP) with human Nav1.4 and Nav1.5 in order to unravel the primary reason for the production of its adverse reactions on the skeletal and cardiac muscles. Our results suggest that the rational distribution of residues with ring structures near position 38 and positive residues in the C-terminal on AGAP are critical factors to ensure its analgesic efficacy. Moreover, the substitution for residues with benzene is beneficial to reduce its side effects.

The voltage-gated sodium channel NaV1.7 is an important target for drug development due to its role in pain perception. Recombinant expression of full-length channels and their use for biophysical characterization of interactions with potential drug candidates is challenging due to the protein size and complexity. To overcome this issue, we developed a protocol for the recombinant expression in E. coli and refolding into lipids of the isolated voltage sensing domain (VSD) of repeat II of NaV1.7, obtaining yields of about 2 mg of refolded VSD from 1 L bacterial cell culture. This VSD is known to be involved in the binding of a number of gating-modifier toxins, including the tarantula toxins ProTx-II and GpTx-I. Binding studies using microscale thermophoresis showed that recombinant refolded VSD binds both of these toxins with dissociation constants in the high nM range, and their relative binding affinities reflect the relative IC50 values of these toxins for full-channel inhibition. Additionally, we expressed mutant VSDs incorporating single amino acid substitutions that had previously been shown to affect the activity of ProTx-II on full channel. We found decreases in GpTx-I binding affinity for these mutants, consistent with a similar binding mechanism for GpTx-I as compared to that of ProTx-II. Therefore, this recombinant VSD captures many of the native interactions between NaV1.7 and tarantula gating-modifier toxins and represents a valuable tool for elucidating details of toxin binding and specificity that could help in the design of non-addictive pain medication acting through NaV1.7 inhibition.

The voltage-gated sodium channel Nav1.8 is linked to neuropathic and inflammatory pain, highlighting the potential to serve as a drug target. However, the biophysical mechanisms that regulate Nav1.8 activation and inactivation gating are not completely understood. Progress has been hindered by a lack of biochemical tools for examining Nav1.8 gating mechanisms. Arizona bark scorpion (Centruroides sculpturatus) venom proteins inhibit Nav1.8 and block pain in grasshopper mice (Onychomys torridus). These proteins provide tools for examining Nav1.8 structure–activity relationships. To identify proteins that inhibit Nav1.8 activity, venom samples were fractioned using liquid chromatography (reversed-phase and ion exchange). A recombinant Nav1.8 clone expressed in ND7/23 cells was used to identify subfractions that inhibited Nav1.8 Na+ current. Mass-spectrometry-based bottom-up proteomic analyses identified unique peptides from inhibitory subfractions. A search of the peptides against the AZ bark scorpion venom gland transcriptome revealed four novel proteins between 40 and 60% conserved with venom proteins from scorpions in four genera (Centruroides, Parabuthus, Androctonus, and Tityus). Ranging from 63 to 82 amino acids, each primary structure includes eight cysteines and a “CXCE” motif, where X = an aromatic residue (tryptophan, tyrosine, or phenylalanine). Electrophysiology data demonstrated that the inhibitory effects of bioactive subfractions can be removed by hyperpolarizing the channels, suggesting that proteins may function as gating modifiers as opposed to pore blockers.

Oxaliplatin is a third-generation platinum-based chemotherapeutic drug widely used in colorectal cancer treatment. Although potent against this tumor, it can induce cold and mechanical allodynia even after a single injection. The currently used drugs to attenuate this allodynia can also cause unwanted effects, which limit their use. Bee venom acupuncture (BVA) is widely used in Korean medicine to treat pain. Although the effect of BVA on oxaliplatin-induced neuropathic pain has been addressed in many studies, its action on dorsal root ganglia (DRG) neurons has never been investigated. A single oxaliplatin injection (6 mg/kg, intraperitoneally) induced cold and mechanical allodynia, and BVA (0.1 and 1 mg/kg, subcutaneous, ST36) dose-dependently decreased allodynia in rats. On acutely dissociated lumbar 4–6 DRG neurons, 10 min application of oxaliplatin (100 μM) shifted the voltage-dependence of sodium conductance toward negative membrane potentials in A- but not C-fibers. The resting membrane potential remained unchanged, but the action potential threshold decreased significantly compared to that of the control (p < 0.05). However, 0.1 μg/mL of BVA administration increased the lowered action potential threshold. In conclusion, these results suggest that BVA may attenuate oxaliplatin-induced neuropathic pain by altering the action potential threshold in A-fiber DRG neurons.

Research in toxinology has created a pharmacological paradox. With an estimated 220,000 venomous animals worldwide, the study of peptidyl toxins provides a vast number of effector molecules. However, due to the complexity of the protein-protein interactions, there are fewer than ten venom-derived molecules on the market. Structural characterization and identification of post-translational modifications are essential to develop biological lead structures into pharmaceuticals. Utilizing advancements in mass spectrometry, we have created a high definition approach that fuses conventional high-resolution MS-MS with ion mobility spectrometry (HDMSE) to elucidate these primary structure characteristics. We investigated venom from ten species of “tiger” spider (Genus: Poecilotheria) and discovered they contain isobaric conformers originating from non-enzymatic Asp isomerization. One conformer pair conserved in five of ten species examined, denominated PcaTX-1a and PcaTX-1b, was found to be a 36-residue peptide with a cysteine knot, an amidated C-terminus, and isoAsp33Asp substitution. Although the isomerization of Asp has been implicated in many pathologies, this is the first characterization of Asp isomerization in a toxin and demonstrates the isomerized product’s diminished physiological effects. This study establishes the value of a HDMSE approach to toxin screening and characterization.

Voltage-gated sodium (NaV) channels are responsible for propagating action potentials in excitable cells. NaV1.7 plays a crucial role in the human pain signalling pathway and it is an important therapeutic target for treatment of chronic pain. Numerous spider venom peptides have been shown to modulate the activity of NaV channels and these peptides represent a rich source of research tools and therapeutic lead molecules. The aim of this study was to determine the diversity of NaV1.7-active peptides in the venom of an Australian Phlogius sp. tarantula and to characterise their potency and subtype selectivity. We isolated three novel peptides, μ-TRTX-Phlo1a, -Phlo1b and -Phlo2a, that inhibit human NaV1.7 (hNaV1.7). Phlo1a and Phlo1b are 35-residue peptides that differ by one amino acid and belong in NaSpTx family 2. The partial sequence of Phlo2a revealed extensive similarity with ProTx-II from NaSpTx family 3. Phlo1a and Phlo1b inhibit hNaV1.7 with IC50 values of 459 and 360 nM, respectively, with only minor inhibitory activity on rat NaV1.2 and hNaV1.5. Although similarly potent at hNaV1.7 (IC50 333 nM), Phlo2a was less selective, as it also potently inhibited rNaV1.2 and hNaV1.5. All three peptides cause a depolarising shift in the voltage-dependence of hNaV1.7 activation.

Voltage-gated sodium channels (NaVs) are a key determinant of neuronal signalling. Neurotoxins from diverse taxa that selectively activate or inhibit NaV channels have helped unravel the role of NaV channels in diseases, including chronic pain. Spider venoms contain the most diverse array of inhibitor cystine knot (ICK) toxins (knottins). This review provides an overview on how spider knottins modulate NaV channels and describes the structural features and molecular determinants that influence their affinity and subtype selectivity. Genetic and functional evidence support a major involvement of NaV subtypes in various chronic pain conditions. The exquisite inhibitory properties of spider knottins over key NaV subtypes make them the best lead molecules for the development of novel analgesics to treat chronic pain.

Voltage-gated sodium channels (VGSC) are the primary mediators of electrical signal amplification and propagation in excitable cells. VGSC subtypes are diverse, with different biophysical and pharmacological properties, and varied tissue distribution. Altered VGSC expression and/or increased VGSC activity in sensory neurons is characteristic of inflammatory and neuropathic pain states. Therefore, VGSC modulators could be used in prospective analgesic compounds. VGSCs have specific binding sites for four conotoxin families: μ-, μO-, δ- and ί-conotoxins. Various studies have identified that the binding site of these peptide toxins is restricted to well-defined areas or domains. To date, only the μ- and μO-family exhibit analgesic properties in animal pain models. This review will focus on conotoxins from the μ- and μO-families that act on neuronal VGSCs. Examples of how these conotoxins target various pharmacologically important neuronal ion channels, as well as potential problems with the development of drugs from conotoxins, will be discussed.

Spider venoms are complex mixtures of biologically active components with potentially interesting applications for drug discovery or for agricultural purposes. The spider Phoneutria nigriventer is responsible for a number of envenomations with sometimes severe clinical manifestations in humans. A more efficient treatment requires a comprehensive knowledge of the venom composition and of the action mechanism of the constituting components. PnTx2-1 (also called δ-ctenitoxin-Pn1a) is a 53-amino-acid-residue peptide isolated from the venom fraction PhTx2. Although PnTx2-1 is classified as a neurotoxin, its molecular target has remained unknown. This study describes the electrophysiological characterization of PnTx2-1 as a modulator of voltage-gated sodium channels. PnTx2-1 is investigated for its activity on seven mammalian NaV-channel isoforms, one insect NaV channel and one arachnid NaV channel. Furthermore, comparison of the activity of both PnTx2-1 and PnTx2-6 on NaV1.5 channels reveals that this family of Phoneutria toxins modulates the cardiac NaV channel in a bifunctional manner, resulting in an alteration of the inactivation process and a reduction of the sodium peak current.

Saxitoxin (STX) and its analogs are paralytic alkaloid neurotoxins that block the voltage-gated sodium channel pore (Nav), impeding passage of Na+ ions into the intracellular space, and thereby preventing the action potential in the peripheral nervous system and skeletal muscle. The marine dinoflagellate Gymnodinium catenatum produces an array of such toxins, including the recently discovered benzoyl analogs, for which the mammalian toxicities are essentially unknown. We subjected STX and its analogs to a theoretical docking simulation based upon two alternative tri-dimensional models of the Nav1.4 to find a relationship between the binding properties and the known mammalian toxicity of selected STX analogs. We inferred hypothetical toxicities for the benzoyl analogs from the modeled values. We demonstrate that these toxins exhibit different binding modes with similar free binding energies and that these alternative binding modes are equally probable. We propose that the principal binding that governs ligand recognition is mediated by electrostatic interactions. Our simulation constitutes the first in silico modeling study on benzoyl-type paralytic toxins and provides an approach towards a better understanding of the mode of action of STX and its analogs. [ABSTRACT FROM AUTHOR]

Haedoxan A (HA) is a major active ingredient in the herbaceous perennial plant lopseed (Phryma leptostachya L.), which is used as a natural insecticide against insect pests in East Asia. Here, we report that HA delayed the decay rate of evoked excitatory junctional potentials (EJPs) and increased the frequency of miniature EJPs (mEJPs) on the Drosophila neuromuscular junction. HA also caused a significant hyperpolarizing shift of the voltage dependence of fast inactivation of insect sodium channels expressed in Xenopus oocytes. Our results suggest that HA acts on both axonal conduction and synaptic transmission, which can serve as a basis for elucidating the mode of action of HA for further designing and developing new effective insecticides. [ABSTRACT FROM AUTHOR]

Saxitoxin (STX) and its analogs are paralytic alkaloid neurotoxins that block the voltage-gated sodium channel pore (Nav), impeding passage of Na+ ions into the intracellular space, and thereby preventing the action potential in the peripheral nervous system and skeletal muscle. The marine dinoflagellate Gymnodinium catenatum produces an array of such toxins, including the recently discovered benzoyl analogs, for which the mammalian toxicities are essentially unknown. We subjected STX and its analogs to a theoretical docking simulation based upon two alternative tri-dimensional models of the Nav1.4 to find a relationship between the binding properties and the known mammalian toxicity of selected STX analogs. We inferred hypothetical toxicities for the benzoyl analogs from the modeled values. We demonstrate that these toxins exhibit different binding modes with similar free binding energies and that these alternative binding modes are equally probable. We propose that the principal binding that governs ligand recognition is mediated by electrostatic interactions. Our simulation constitutes the first in silico modeling study on benzoyl-type paralytic toxins and provides an approach towards a better understanding of the mode of action of STX and its analogs.

Voltage-gated sodium channels (VGSCs, or Na v ) are important determinants of action potential generation and propagation. Efforts are underway to develop medicines targeting different channel subtypes for the treatment of related channelopathies. However, a high degree of conservation across its nine subtypes could lead to the off-target adverse effects on skeletal and cardiac muscles due to acting on primary skeletal muscle sodium channel Na v 1.4 and cardiac muscle sodium channel Na v 1.5, respectively. For a long evolutionary process, some peptide toxins from venoms have been found to be highly potent yet selective on ion channel subtypes and, therefore, hold the promising potential to be developed into therapeutic agents. In this research, all-atom molecular dynamic methods were used to elucidate the selective mechanisms of an analgesic-antitumor β-scorpion toxin (AGAP) with human Na v 1.4 and Na v 1.5 in order to unravel the primary reason for the production of its adverse reactions on the skeletal and cardiac muscles. Our results suggest that the rational distribution of residues with ring structures near position 38 and positive residues in the C-terminal on AGAP are critical factors to ensure its analgesic efficacy. Moreover, the substitution for residues with benzene is beneficial to reduce its side effects.

Voltage-gated sodium channels (VGSC) are the primary mediators of electrical signal amplification and propagation in excitable cells. VGSC subtypes are diverse, with different biophysical and pharmacological properties, and varied tissue distribution. Altered VGSC expression and/or increased VGSC activity in sensory neurons is characteristic of inflammatory and neuropathic pain states. Therefore, VGSC modulators could be used in prospective analgesic compounds. VGSCs have specific binding sites for four conotoxin families: μ-, μO-, δ- and í-conotoxins. Various studies have identified that the binding site of these peptide toxins is restricted to well-defined areas or domains. To date, only the μ- and μO-family exhibit analgesic properties in animal pain models. This review will focus on conotoxins from the μ- and μO-families that act on neuronal VGSCs. Examples of how these conotoxins target various pharmacologically important neuronal ion channels, as well as potential problems with the development of drugs from conotoxins, will be discussed. [ABSTRACT FROM AUTHOR]

The voltage-gated sodium channel NaV1.7 is an important target for drug development due to its role in pain perception. Recombinant expression of full-length channels and their use for biophysical characterization of interactions with potential drug candidates is challenging due to the protein size and complexity. To overcome this issue, we developed a protocol for the recombinant expression in E. coli and refolding into lipids of the isolated voltage sensing domain (VSD) of repeat II of NaV1.7, obtaining yields of about 2 mg of refolded VSD from 1 L bacterial cell culture. This VSD is known to be involved in the binding of a number of gating-modifier toxins, including the tarantula toxins ProTx-II and GpTx-I. Binding studies using microscale thermophoresis showed that recombinant refolded VSD binds both of these toxins with dissociation constants in the high nM range, and their relative binding affinities reflect the relative IC50 values of these toxins for full-channel inhibition. Additionally, we expressed mutant VSDs incorporating single amino acid substitutions that had previously been shown to affect the activity of ProTx-II on full channel. We found decreases in GpTx-I binding affinity for these mutants, consistent with a similar binding mechanism for GpTx-I as compared to that of ProTx-II. Therefore, this recombinant VSD captures many of the native interactions between NaV1.7 and tarantula gating-modifier toxins and represents a valuable tool for elucidating details of toxin binding and specificity that could help in the design of non-addictive pain medication acting through NaV1.7 inhibition. [ABSTRACT FROM AUTHOR]

The voltage-gated sodium channel Nav1.8 is linked to neuropathic and inflammatory pain, highlighting the potential to serve as a drug target. However, the biophysical mechanisms that regulate Nav1.8 activation and inactivation gating are not completely understood. Progress has been hindered by a lack of biochemical tools for examining Nav1.8 gating mechanisms. Arizona bark scorpion (Centruroides sculpturatus) venom proteins inhibit Nav1.8 and block pain in grasshopper mice (Onychomys torridus). These proteins provide tools for examining Nav1.8 structure–activity relationships. To identify proteins that inhibit Nav1.8 activity, venom samples were fractioned using liquid chromatography (reversed-phase and ion exchange). A recombinant Nav1.8 clone expressed in ND7/23 cells was used to identify subfractions that inhibited Nav1.8 Na+ current. Mass-spectrometry-based bottom-up proteomic analyses identified unique peptides from inhibitory subfractions. A search of the peptides against the AZ bark scorpion venom gland transcriptome revealed four novel proteins between 40 and 60% conserved with venom proteins from scorpions in four genera (Centruroides, Parabuthus, Androctonus, and Tityus). Ranging from 63 to 82 amino acids, each primary structure includes eight cysteines and a "CXCE" motif, where X = an aromatic residue (tryptophan, tyrosine, or phenylalanine). Electrophysiology data demonstrated that the inhibitory effects of bioactive subfractions can be removed by hyperpolarizing the channels, suggesting that proteins may function as gating modifiers as opposed to pore blockers. [ABSTRACT FROM AUTHOR]

Oxaliplatin is a third-generation platinum-based chemotherapeutic drug widely used in colorectal cancer treatment. Although potent against this tumor, it can induce cold and mechanical allodynia even after a single injection. The currently used drugs to attenuate this allodynia can also cause unwanted effects, which limit their use. Bee venom acupuncture (BVA) is widely used in Korean medicine to treat pain. Although the effect of BVA on oxaliplatin-induced neuropathic pain has been addressed in many studies, its action on dorsal root ganglia (DRG) neurons has never been investigated. A single oxaliplatin injection (6 mg/kg, intraperitoneally) induced cold and mechanical allodynia, and BVA (0.1 and 1 mg/kg, subcutaneous, ST36) dose-dependently decreased allodynia in rats. On acutely dissociated lumbar 4–6 DRG neurons, 10 min application of oxaliplatin (100 μM) shifted the voltage-dependence of sodium conductance toward negative membrane potentials in A- but not C-fibers. The resting membrane potential remained unchanged, but the action potential threshold decreased significantly compared to that of the control (p < 0.05). However, 0.1 μg/mL of BVA administration increased the lowered action potential threshold. In conclusion, these results suggest that BVA may attenuate oxaliplatin-induced neuropathic pain by altering the action potential threshold in A-fiber DRG neurons. [ABSTRACT FROM AUTHOR]

Research in toxinology has created a pharmacological paradox. With an estimated 220,000 venomous animals worldwide, the study of peptidyl toxins provides a vast number of effector molecules. However, due to the complexity of the protein-protein interactions, there are fewer than ten venom-derived molecules on the market. Structural characterization and identification of post-translational modifications are essential to develop biological lead structures into pharmaceuticals. Utilizing advancements in mass spectrometry, we have created a high definition approach that fuses conventional high-resolution MS-MS with ion mobility spectrometry (HDMSE) to elucidate these primary structure characteristics. We investigated venom from ten species of "tiger" spider (Genus: Poecilotheria) and discovered they contain isobaric conformers originating from non-enzymatic Asp isomerization. One conformer pair conserved in five of ten species examined, denominated PcaTX-1a and PcaTX-1b, was found to be a 36-residue peptide with a cysteine knot, an amidated C-terminus, and isoAsp33Asp substitution. Although the isomerization of Asp has been implicated in many pathologies, this is the first characterization of Asp isomerization in a toxin and demonstrates the isomerized product's diminished physiological effects. This study establishes the value of a HDMSE approach to toxin screening and characterization. [ABSTRACT FROM AUTHOR]

Voltage-gated sodium channels (NaVs) are a key determinant of neuronal signalling. Neurotoxins from diverse taxa that selectively activate or inhibit NaV channels have helped unravel the role of NaV channels in diseases, including chronic pain. Spider venoms contain the most diverse array of inhibitor cystine knot (ICK) toxins (knottins). This review provides an overview on how spider knottins modulate NaV channels and describes the structural features and molecular determinants that influence their affinity and subtype selectivity. Genetic and functional evidence support a major involvement of NaV subtypes in various chronic pain conditions. The exquisite inhibitory properties of spider knottins over key NaV subtypes make them the best lead molecules for the development of novel analgesics to treat chronic pain. [ABSTRACT FROM AUTHOR]

Spider venoms are complex mixtures of biologically active components with potentially interesting applications for drug discovery or for agricultural purposes. The spider Phoneutria nigriventer is responsible for a number of envenomations with sometimes severe clinical manifestations in humans. A more efficient treatment requires a comprehensive knowledge of the venom composition and of the action mechanism of the constituting components. PnTx2-1 (also called δ-ctenitoxin-Pn1a) is a 53-amino-acid-residue peptide isolated from the venom fraction PhTx2. Although PnTx2-1 is classified as a neurotoxin, its molecular target has remained unknown. This study describes the electrophysiological characterization of PnTx2-1 as a modulator of voltage-gated sodium channels. PnTx2-1 is investigated for its activity on seven mammalian NaV-channel isoforms, one insect NaV channel and one arachnid NaV channel. Furthermore, comparison of the activity of both PnTx2-1 and PnTx2-6 on NaV1.5 channels reveals that this family of Phoneutria toxins modulates the cardiac NaV channel in a bifunctional manner, resulting in an alteration of the inactivation process and a reduction of the sodium peak current. [ABSTRACT FROM AUTHOR]

Saxitoxin (STX) and its analogs are paralytic alkaloid neurotoxins that block the voltage-gated sodium channel pore (Nav), impeding passage of Na⁺ ions into the intracellular space, and thereby preventing the action potential in the peripheral nervous system and skeletal muscle. The marine dinoflagellate Gymnodinium catenatum produces an array of such toxins, including the recently discovered benzoyl analogs, for which the mammalian toxicities are essentially unknown. We subjected STX and its analogs to a theoretical docking simulation based upon two alternative tri-dimensional models of the Nav1.4 to find a relationship between the binding properties and the known mammalian toxicity of selected STX analogs. We inferred hypothetical toxicities for the benzoyl analogs from the modeled values. We demonstrate that these toxins exhibit different binding modes with similar free binding energies and that these alternative binding modes are equally probable. We propose that the principal binding that governs ligand recognition is mediated by electrostatic interactions. Our simulation constitutes the first in silico modeling study on benzoyl-type paralytic toxins and provides an approach towards a better understanding of the mode of action of STX and its analogs.

Haedoxan A (HA) is a major active ingredient in the herbaceous perennial plant lopseed (Phryma leptostachya L.), which is used as a natural insecticide against insect pests in East Asia. Here, we report that HA delayed the decay rate of evoked excitatory junctional potentials (EJPs) and increased the frequency of miniature EJPs (mEJPs) on the Drosophila neuromuscular junction. HA also caused a significant hyperpolarizing shift of the voltage dependence of fast inactivation of insect sodium channels expressed in Xenopus oocytes. Our results suggest that HA acts on both axonal conduction and synaptic transmission, which can serve as a basis for elucidating the mode of action of HA for further designing and developing new effective insecticides.

Voltage-gated sodium (NaV) channels are responsible for propagating action potentials in excitable cells. NaV1.7 plays a crucial role in the human pain signalling pathway and it is an important therapeutic target for treatment of chronic pain. Numerous spider venom peptides have been shown to modulate the activity of NaV channels and these peptides represent a rich source of research tools and therapeutic lead molecules. The aim of this study was to determine the diversity of NaV1.7-active peptides in the venom of an Australian Phlogius sp. tarantula and to characterise their potency and subtype selectivity. We isolated three novel peptides, μ-TRTX-Phlo1a, -Phlo1b and -Phlo2a, that inhibit human NaV1.7 (hNaV1.7). Phlo1a and Phlo1b are 35-residue peptides that differ by one amino acid and belong in NaSpTx family 2. The partial sequence of Phlo2a revealed extensive similarity with ProTx-II from NaSpTx family 3. Phlo1a and Phlo1b inhibit hNaV1.7 with IC50 values of 459 and 360 nM, respectively, with only minor inhibitory activity on rat NaV1.2 and hNaV1.5. Although similarly potent at hNaV1.7 (IC50 333 nM), Phlo2a was less selective, as it also potently inhibited rNaV1.2 and hNaV1.5. All three peptides cause a depolarising shift in the voltage-dependence of hNaV1.7 activation.