From foe to friend: using animal toxins to investigate ion channel function

Ion channels are vital contributors to cellular communication in a wide range of organisms, a distinct feature that renders this ubiquitous family of membrane-spanning proteins a prime target for toxins found in animal venom. For many years, the unique properties of these naturally-occurring molecules have enabled researchers to probe the structural and functional features of ion channels and to define their physiological roles in normal and diseased tissues. To illustrate their considerable impact on the ion channel field, this review will highlight fundamental insights into toxin-channel interactions as well as recently developed toxin screening methods and practical applications of engineered toxins

Rational engineering defines a molecular switch that is essential for activity of spider-venom peptides against the analgesics target na(v)1.7

Many spider-venom peptides are known to modulate the activity of the voltage-gated sodium (NaV) subtype 1.7 (NaV1.7) channel, which has emerged as a promising analgesic target. In particular, a class of spider-venom peptides (NaSpTx1) has been found to potently inhibit NaV1.7 (nanomolar IC), and has been shown to produce analgesic effects in animals. However, onemember of this family [μ-TRTX-Hhn2b (Hhn2b)] does not inhibit mammalian NaV channels expressed in dorsal root ganglia at concentrations up to 100 μM. This peptide is classified as a NaSpTx1 member by virtue of its cysteine spacing and sequence conservation over functionally important residues. Here, we have performed detailed structural and functional analyses of Hhn2b, leading us to identify two nonpharmacophore residues that contribute to human NaV1.7 (hNaV1.7) inhibition by nonoverlapping mechanisms. These findings allowed us to produce a double mutant of Hhn2b that shows nanomolar inhibition of hNaV1.7. Traditional structure/function analysis did not provide sufficient resolution to identify the mechanism underlying the observed gain of function. However, by solving the high-resolution structure of both the wild-type and mutant peptides using advanced multidimensional NMR experiments, we were able to uncover a previously unknown network of interactions that stabilize the pharmacophore region of this class of venom peptides. We further monitored the lipid binding properties of the peptides and identified that one of the key amino acid substitutions also selectively modulates the binding of the peptide to anionic lipids. These results will further aid the development of peptide-based analgesics for the treatment of chronic pain

Rational Engineering Defines a Molecular Switch That Is Essential for Activity of Spider-Venom Peptides against the Analgesics Target Na V 1.7 s

ABSTRACT Many spider-venom peptides are known to modulate the activity of the voltage-gated sodium (Na V ) subtype 1.7 (Na V 1.7) channel, which has emerged as a promising analgesic target. In particular, a class of spider-venom peptides (NaSpTx1) has been found to potently inhibit Na V 1.7 (nanomolar IC 50 ), and has been shown to produce analgesic effects in animals. However, one member of this family [m-TRTX-Hhn2b (Hhn2b)] does not inhibit mammalian Na V channels expressed in dorsal root ganglia at concentrations up to 100 mM. This peptide is classified as a NaSpTx1 member by virtue of its cysteine spacing and sequence conservation over functionally important residues. Here, we have performed detailed structural and functional analyses of Hhn2b, leading us to identify two nonpharmacophore residues that contribute to human Na V 1.7 (hNa V 1.7) inhibition by nonoverlapping mechanisms. These findings allowed us to produce a double mutant of Hhn2b that shows nanomolar inhibition of hNa V 1.7. Traditional structure/function analysis did not provide sufficient resolution to identify the mechanism underlying the observed gain of function. However, by solving the high-resolution structure of both the wild-type and mutant peptides using advanced multidimensional NMR experiments, we were able to uncover a previously unknown network of interactions that stabilize the pharmacophore region of this class of venom peptides. We further monitored the lipid binding properties of the peptides and identified that one of the key amino acid substitutions also selectively modulates the binding of the peptide to anionic lipids. These results will further aid the development of peptide-based analgesics for the treatment of chronic pain

Spider-venom peptides that target voltage-gated sodium channels: pharmacological tools and potential therapeutic leads

Voltage-gated sodium (Na-v) channels play a central role in the propagation of action potentials in excitable cells in both humans and insects. Many venomous animals have therefore evolved toxins that modulate the activity of Na-v channels in order to subdue their prey and deter predators. Spider venoms in particular are rich in Na-v channel modulators, with one-third of all known ion channel toxins from spider venoms acting on Na-v channels. Here we review the landscape of spider-venom peptides that have so far been described to target vertebrate or invertebrate Na-v channels. These peptides fall into 12 distinct families based on their primary structure and cysteine scaffold. Some of these peptides have become useful pharmacological tools, while others have potential as therapeutic leads because they target specific Na-v channel subtypes that are considered to be important analgesic targets. Spider venoms are conservatively predicted to contain more than 10 million bioactive peptides and so far only 0.01% of this diversity been characterised. Thus, it is likely that future research will reveal additional structural classes of spider-venom peptides that target Na-v channels. (c) 2012 Elsevier Ltd. All rights reserved

Isolation, synthesis and characterization of omega-TRTX-Cc1a, a novel tarantula venom peptide that selectively targets L-type CaV channels

Spider venoms are replete with peptidic ion channel modulators, often with novel subtype selectivity, making them a rich source of pharmacological tools and drug leads. In a search for subtype-selective blockers of voltage-gated calcium (Ca-v) channels, we isolated and characterized a novel 39-residue peptide, omega-TRTX-Cc1a (Cc1a), from the venom of the tarantula Citharischius crawshayi (now Pelinobius muticus). Cc1a is 67% identical to the spider toxin omega-TRTX-Hg1a, an inhibitor of Ca(v)2.3 channels. We assembled Cc1a using a combination of Boc solid-phase peptide synthesis and native chemical ligation. Oxidative folding yielded two stable, slowly interconverting isomers. Cc1a preferentially inhibited Ba2+ currents (I-Ba) mediated by L-type (Ca(v)1.2 and Ca(v)1.3) Ca-v channels heterologously expressed in Xenopus oocytes, with half-maximal inhibitory concentration (IC50) values of 825 nM and 2.24 mu M respectively. In rat dorsal root ganglion neurons, Cc1a inhibited I-Ba mediated by high voltage-activated Ca-v channels but did not affect low voltage-activated T-type Ca-v channels. Cc1a exhibited weak activity at Na(v)1.5 and Na(v)1.7 voltage-gated sodium (Na-v) channels stably expressed in mammalian HEK or CHO cells, respectively. Experiments with modified Cc1a peptides, truncated at the N-terminus (Delta G1-E5) or C-terminus (Delta W35-V39), demonstrated that the N- and C-termini are important for voltage-gated ion channel modulation. We conclude that Cc1a represents a novel pharmacological tool for probing the structure and function of L-type Cav channels. (C) 2014 Elsevier Inc. All rights reserved

Isolation, synthesis and characterization of ω-TRTX-Cc1a, a novel tarantula venom peptide that selectively targets L-type CaV channels

Spider venoms are replete with peptidic ion channel modulators, often with novel subtype selectivity, making them a rich source of pharmacological tools and drug leads. In a search for subtype-selective blockers of voltage-gated calcium (CaV) channels, we isolated and characterized a novel 39-residue peptide, ω-TRTX-Cc1a (Cc1a), from the venom of the tarantula Citharischius crawshayi (now Pelinobius muticus). Cc1a is 67% identical to the spider toxin ω-TRTX-Hg1a, an inhibitor of CaV2.3 channels. We assembled Cc1a using a combination of Boc solid-phase peptide synthesis and native chemical ligation. Oxidative folding yielded two stable, slowly interconverting isomers. Cc1a preferentially inhibited Ba2+ currents (IBa) mediated by L-type (CaV1.2 and CaV1.3) CaV channels heterologously expressed in Xenopus oocytes, with half-maximal inhibitory concentration (IC50) values of 825 nM and 2.24 μM, respectively. In rat dorsal root ganglion neurons, Cc1a inhibited IBa mediated by high voltage-activated CaV channels but did not affect low voltage-activated T-type CaV channels. Cc1a exhibited weak activity at NaV1.5 and NaV1.7 voltage-gated sodium (NaV) channels stably expressed in mammalian HEK or CHO cells, respectively. Experiments with modified Cc1a peptides, truncated at the N-terminus (ΔG1-E5) or C-terminus (ΔW35-V39), demonstrated that the N- and C-termini are important for voltage-gated ion channel modulation. We conclude that Cc1a represents a novel pharmacological tool for probing the structure and function of L-type CaV channels

Discovery of a selective Na(V)1.7 inhibitor from centipede venom with analgesic efficacy exceeding morphine in rodent pain models

Loss-of-function mutations in the human voltage-gated sodium channel NaV1.7 result in a congenital indifference to pain. Selective inhibitors of NaV1.7 are therefore likely to be powerful analgesics for treating a broad range of pain conditions. Herein we describe the identification of μ-SLPTX-Ssm6a, a unique 46-residue peptide from centipede venom that potently inhibits NaV1.7 with an IC50 of ∼25 nM. μ-SLPTX-Ssm6a has more than 150-fold selectivity for Na V1.7 over all other human NaV subtypes, with the exception of NaV1.2, for which the selectivity is 32-fold. μ-SLPTX-Ssm6a contains three disulfide bonds with a unique connectivity pattern, and it has no significant sequence homology with any previously characterized peptide or protein. μ-SLPTX-Ssm6a proved to be a more potent analgesic than morphine in a rodent model of chemicalinduced pain, and it was equipotent with morphine in rodent models of thermal and acid-induced pain. This study establishes μ-SPTX-Ssm6a as a promising lead molecule for the development of novel analgesics targeting NaV1.7, which might be suitable for treating a wide range of human pain pathologies

Fluorescence labeling of a NaV1.7-targeted peptide for near-infrared nerve visualization

Background: Accidental peripheral nerve injury during surgical intervention results in a broad spectrum of potentially debilitating side effects. Tissue distortion and poor visibility can significantly increase the risk of nerve injury with long-lasting consequences for the patient. We developed and characterized Hs1a-FL, a fluorescent near-infrared molecule for nerve visualization in the operating theater with the aim of helping physicians to visualize nerves during surgery. Hs1a was derived from the venom of the Chinese bird spider, Haplopelma schmidti, and conjugated to Cy7.5 dye. Hs1a-FL was injected intravenously in mice, and harvested nerves were imaged microscopically and with epifluorescence. Results: Hs1a-FL showed specific and stable binding to the sodium channel Na1.7, present on the surface of human and mouse nerves. Hs1a-FL allowed epifluorescence visualization of sciatic mouse nerves with favorable nerve-to-muscle contrast. Conclusions: Fluorescent Na1.7-targeted tracers have the potential to be adopted clinically for the intraoperative visualization of peripheral nerves during surgery, providing guidance for the surgeon and potentially improving the standard of care

Effect of buffer, temperature, and pH on 2D ¹H-¹⁵N HSQC NMR spectra of a disulfide-rich venom peptide (Step 9).

<p>(<b>A</b>) Overlays of the downfield region of 2D ¹H-¹⁵N HSQC spectra of a spider-venom peptide (46 residues, 4 disulfide bonds) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063865#pone.0063865-Lipkin1" target="_blank">[90]</a> acquired at 25°C using different buffers and pH: 20 mM MES pH 6 (pink); sodium phosphate, pH 6 (purple); 20 mM sodium acetate, pH 5 (red); 20 mM sodium citrate, pH 4 (blue). This region of the spectrum shows the sidechain ¹H-¹⁵N correlation for the single Trp residue in this peptide. (<b>B</b>) Effect of temperature on the same resonance. Spectra were acquired in 20 mM citrate, pH 4 at the following temperatures: 10°C (purple); 25°C (red); 40°C (blue). At low pH and high temperature the equilibrium is shifted towards a single conformer, compared to the three conformers apparent at lower temperature.</p