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1.  A distinct sodium channel voltage-sensor locus determines insect selectivity of the spider toxin Dc1a 
Nature communications  2014;5:4350.
β-Diguetoxin-Dc1a (Dc1a) is a toxin from the desert bush spider Diguetia canities that incapacitates insects at concentrations that are non-toxic to mammals. Dc1a promotes opening of German cockroach voltage-gated sodium (Nav) channels (BgNav1), whereas human Nav channels are insensitive. Here, by transplanting commonly targeted S3b-S4 paddle motifs within BgNav1 voltage sensors into Kv2.1, we find that Dc1a interacts with the domain II voltage sensor. In contrast, Dc1a has little effect on sodium currents mediated by PaNav1 channels from the American cockroach even though their domain II paddle motifs are identical. When exploring regions responsible for PaNav1 resistance to Dc1a, we identified two residues within the BgNav1 domain II S1–S2 loop that when mutated to their PaNav1 counterparts drastically reduce toxin susceptibility. Overall, our results reveal a distinct region within insect Nav channels that helps determine Dc1a sensitivity, aconcept that will be valuable for the design of insect-selective insecticides.
PMCID: PMC4115291  PMID: 25014760
voltage-gated sodium channel; voltage sensor; spider toxin; Dc1a; insect; cockroach
2.  The insecticidal neurotoxin Aps III is an atypical knottin peptide that potently blocks insect voltage-gated sodium channels 
Biochemical pharmacology  2013;85(10):10.1016/j.bcp.2013.02.030.
One of the most potent insecticidal venom peptides described to date is Aps III from the venom of the trapdoor spider Apomastus schlingeri. Aps III is highly neurotoxic to lepidopteran crop pests, making it a promising candidate for bioinsecticide development. However, its disulfide-connectivity, three-dimensional structure, and mode of action have not been determined. Here we show that recombinant Aps III (rAps III) is an atypical knottin peptide; three of the disulfide bridges form a classical inhibitor cystine knot motif while the fourth disulfide acts as a molecular staple that restricts the flexibility of an unusually large β hairpin loop that often houses the pharmacophore in this class of toxins. We demonstrate that the irreversible paralysis induced in insects by rAps III results from a potent block of insect voltage-gated sodium channels. Channel block by rAps III is voltage-independent insofar as it occurs without significant alteration in the voltage-dependence of channel activation or steady-state inactivation. Thus, rAps III appears to be a pore blocker that plugs the outer vestibule of insect voltage-gated sodium channels. This mechanism of action contrasts strikingly with virtually all other sodium channel modulators isolated from spider venoms that act as gating modifiers by interacting with one or more of the four voltage-sensing domains of the channel.
PMCID: PMC3654253  PMID: 23473802
voltage-gated sodium channel; neurotoxin; spider-venom peptide; pore blocker; gating modifier; inhibitor cystine knot
3.  Animal Toxins Can Alter the Function of Nav1.8 and Nav1.9 
Toxins  2012;4(8):620-632.
Human voltage-activated sodium (Nav) channels are adept at rapidly transmitting electrical signals across long distances in various excitable tissues. As such, they are amongst the most widely targeted ion channels by drugs and animal toxins. Of the nine isoforms, Nav1.8 and Nav1.9 are preferentially expressed in DRG neurons where they are thought to play an important role in pain signaling. Although the functional properties of Nav1.8 have been relatively well characterized, difficulties with expressing Nav1.9 in established heterologous systems limit our understanding of the gating properties and toxin pharmacology of this particular isoform. This review summarizes our current knowledge of the role of Nav1.8 and Nav1.9 in pain perception and elaborates on the approaches used to identify molecules capable of influencing their function.
PMCID: PMC3446747  PMID: 23012651
Nav1.8; Nav1.9; pain; animal toxins; voltage sensor; voltage-activated sodium channel
4.  Functional properties and toxin pharmacology of a dorsal root ganglion sodium channel viewed through its voltage sensors 
The voltage-activated sodium (Nav) channel Nav1.9 is expressed in dorsal root ganglion (DRG) neurons where it is believed to play an important role in nociception. Progress in revealing the functional properties and pharmacological sensitivities of this non-canonical Nav channel has been slow because attempts to express this channel in a heterologous expression system have been unsuccessful. Here, we use a protein engineering approach to dissect the contributions of the four Nav1.9 voltage sensors to channel function and pharmacology. We define individual S3b–S4 paddle motifs within each voltage sensor, and show that they can sense changes in membrane voltage and drive voltage sensor activation when transplanted into voltage-activated potassium channels. We also find that the paddle motifs in Nav1.9 are targeted by animal toxins, and that these toxins alter Nav1.9-mediated currents in DRG neurons. Our results demonstrate that slowly activating and inactivating Nav1.9 channels have functional and pharmacological properties in common with canonical Nav channels, but also show distinctive pharmacological sensitivities that can potentially be exploited for developing novel treatments for pain.
PMCID: PMC3135324  PMID: 21670206
5.  Targeting sodium channel voltage sensors with spider toxins 
Voltage-activated sodium (Nav) channels are essential in generating and propagating nerve impulses, placing them amongst the most widely targeted ion channels by toxins from venomous organisms. An increasing number of spider toxins have been shown to interfere with the voltage-driven activation process of mammalian Nav channels, possibly by interacting with one or more of their voltage sensors. This review focuses on our existing knowledge of the mechanism by which spider toxins affect Nav channel gating and the possible applications of these toxins in the drug discovery process.
PMCID: PMC2847040  PMID: 20097434
6.  Interactions between lipids and voltage sensor paddles detected with tarantula toxins 
Nature structural & molecular biology  2009;16(10):1080-1085.
Voltage-activated ion channels open and close in response to changes in voltage, a property that is essential for generating nerve impulses. Studies on voltage-activated potassium (Kv) channels show that voltage-sensor activation is sensitive to the composition of lipids in the surrounding membrane. Here we explore the interaction of lipids with S1–S4 voltage-sensing domains, and find that the conversion of the membrane lipid sphingomyelin to ceramide-1-phosphate alters voltage-sensor activation in an S1–S4 voltage-sensing protein lacking an associated pore domain, and that the S3b–S4 paddle motif determines the effects of lipid modification on Kv channels. Using tarantula toxins that bind to paddle motifs within the membrane, we identify mutations in the paddle motif that weaken toxin binding by disrupting lipid-paddle interactions. Our results suggest that lipids bind to voltage-sensing domains and demonstrate that the pharmacological sensitivities of voltage-activated ion channels are influenced by the surrounding lipid membrane.
PMCID: PMC2782670  PMID: 19783984
7.  Deconstructing voltage sensor function and pharmacology in sodium channels 
Nature  2008;456(7219):202-208.
Voltage-activated sodium (Nav) channels are crucial for the generation and propagation of nerve impulses, and as such are amongst the most widely targeted ion channels by toxins and drugs. The four voltage sensors in Nav channels have distinct amino acid sequences, raising fundamental questions about their relative contributions to the function and pharmacology of the channel. Here we use four-fold symmetric voltage-activated potassium (Kv) channels as reporters to examine the contributions of individual Nav channel S3b-S4 paddle motifs to the kinetics of voltage sensor activation and to forming toxin receptors. Our results uncover binding sites for toxins from tarantula and scorpion venom on each of the four paddle motifs in Nav channels and reveal how paddle-specific interactions can be used to reshape Nav channel activity. One paddle motif is unique in that it slows voltage sensor activation and toxins selectively targeting this motif impede Nav channel inactivation. This reporter approach and the principles that emerge will be useful in developing new drugs for treating pain and Nav channelopathies.
PMCID: PMC2587061  PMID: 19005548
8.  Sea anemone venom as a source of insecticidal peptides acting on voltage-gated Na+ channels 
Sea anemones produce a myriad of toxic peptides and proteins of which a large group acts on voltage-gated Na+ channels. However, in comparison to other organisms, their venoms and toxins are poorly studied. Most of the known voltage-gated Na+ channel toxins isolated from sea anemone venoms act on neurotoxin receptor site 3 and inhibit the inactivation of these channels. Furthermore, it seems that most of these toxins have a distinct preference for crustaceans. Given the close evolutionary relationship between crustaceans and insects, it is not surprising that sea anemone toxins also profoundly affect insect voltage-gated Na+ channels, which constitutes the scope of this review. For this reason, these peptides can be considered as insecticidal lead compounds in the development of insecticides.
PMCID: PMC1868498  PMID: 17224168
9.  Differential effects of five ‘classical’ scorpion β-toxins on rNav1.2a and DmNav1 provide clues on species-selectivity 
In general, scorpion β-toxins have been well examined. However, few in-depth studies have been devoted to species selectivity and affinity comparisons on the different voltage-activated Na+ channels since they have become available as cloned channels that can be studied in heterologous expression systems. As a result, their classification is largely historical and dates from early in vivo experiments on mice and cockroach and fly larvae.
In this study, we aimed to provide an updated overview of selectivity and affinity of scorpion β-toxins towards voltage-activated Na+ channels of vertebrates or invertebrates. As pharmacological tools, we used the classic β-toxins AaHIT, Css II, Css IV, Css VI and Ts VII and tested them on the neuronal vertebrate voltage-activated Na+ channel, rNav1.2a. For comparison, its invertebrate counterpart, DmNav1, was also tested. Both these channels were expressed in Xenopus laevis oocytes and the currents measured with the two-electrode voltage-clamp technique. We supplemented this data with several binding displacement studies on rat brain synaptosomes. The results lead us to propose a general classification and a novel nomenclature of scorpion β-toxins based on pharmacological activity.
PMCID: PMC1868420  PMID: 17118417
scorpion β-toxins; voltage-activated Na+ channels; species-selectivity; Centruroides suffusus suffusus; Androctonus australis Hector
10.  Voltage-gated sodium channel modulation by scorpion α-toxins 
Voltage-gated Na+ channels are integral membrane proteins that function as a gateway for a selective permeation of sodium ions across biological membranes. In this way, they are crucial players for the generation of action potentials in excitable cells. Voltage-gated Na+ channels are encoded by at least nine genes in mammals. The different isoforms have remarkably similar functional properties, but small changes in function and pharmacology are biologically well-defined, as underscored by mutations that cause several diseases and by modulation of a myriad of compounds respectively. This review will stress on the modulation of voltage-gated Na+ channels by scorpion alpha-toxins. Nature has designed these two classes of molecules as if they were predestined to each other: an inevitable ‘encounter’ between a voltage-gated Na+ channel isoform and an alpha-toxin from scorpion venom indeed results in a dramatically changed Na+ current phenotype with clear-cut consequences on electrical excitability and sometimes life or death. This fascinating aspect justifies an overview on scorpion venoms, their alpha-toxins and the Na+ channel targets they are built for, as well as on the molecular determinants that govern the selectivity and affinity of this ‘inseparable duo’.
PMCID: PMC1808227  PMID: 17087986

Results 1-10 (10)