A distinct sodium channel voltage-sensor locus determines insect selectivity of the spider toxin Dc1a

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.

[1]  G. King,et al.  A rational nomenclature for naming peptide toxins from spiders and other venomous animals. , 2008, Toxicon : official journal of the International Society on Toxinology.

[2]  B. Bean,et al.  Functional properties and toxin pharmacology of a dorsal root ganglion sodium channel viewed through its voltage sensors , 2011, The Journal of general physiology.

[3]  Ron O. Dror,et al.  Mechanism of Voltage Gating in Potassium Channels , 2012, Science.

[4]  K. Swartz,et al.  Tarantula toxins interacting with voltage sensors in potassium channels. , 2007, Toxicon : official journal of the International Society on Toxinology.

[5]  W. Catterall,et al.  Structure and function of the voltage sensor of sodium channels probed by a beta-scorpion toxin. , 2006, The Journal of biological chemistry.

[6]  Francisco Bezanilla,et al.  Voltage-Sensing Residues in the S2 and S4 Segments of the Shaker K+ Channel , 1996, Neuron.

[7]  David L. Worcester,et al.  Structure and hydration of membranes embedded with voltage-sensing domains , 2009, Nature.

[8]  Xiao Tao,et al.  A Gating Charge Transfer Center in Voltage Sensors , 2010, Science.

[9]  J. Vobecký,et al.  Tarantula Toxins Interact with Voltage Sensors within Lipid Membranes , 2007, The Journal of general physiology.

[10]  Zhiqi Liu,et al.  Persistent tetrodotoxin-sensitive sodium current resulting from U-to-C RNA editing of an insect sodium channel. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[11]  Sophie Quinchard,et al.  The discovery of a novel sodium channel in the cockroach Periplaneta americana: evidence for an early duplication of the para-like gene. , 2009, Insect biochemistry and molecular biology.

[12]  Shengjie Li,et al.  Recent Advances , 2018, Journal of Optimization Theory and Applications.

[13]  K. Swartz,et al.  Deconstructing voltage sensor function and pharmacology in sodium channels , 2008, Nature.

[14]  T Hoshi,et al.  Shaker potassium channel gating. III: Evaluation of kinetic models for activation , 1994, The Journal of general physiology.

[15]  D. M. Soderlund,et al.  Molecular mechanisms of pyrethroid insecticide neurotoxicity: recent advances , 2011, Archives of Toxicology.

[16]  G. King,et al.  Spider-venom peptides: structure, pharmacology, and potential for control of insect pests. , 2013, Annual review of entomology.

[17]  A. L. Goldin,et al.  Substitutions in the Domain III Voltage-sensing Module Enhance the Sensitivity of an Insect Sodium Channel to a Scorpion β-Toxin* , 2011, The Journal of Biological Chemistry.

[18]  W. Hodgson,et al.  Neurotoxic and insecticidal properties of venom from the Australian theraphosid spider Selenotholus foelschei. , 2008, Neurotoxicology.

[19]  D. M. Soderlund,et al.  Divergent actions of the pyrethroid insecticides S-bioallethrin, tefluthrin, and deltamethrin on rat Na(v)1.6 sodium channels. , 2010, Toxicology and applied pharmacology.

[20]  James O. Jackson,et al.  Common Molecular Determinants of Tarantula Huwentoxin-IV Inhibition of Na+ Channel Voltage Sensors in Domains II and IV* , 2011, The Journal of Biological Chemistry.

[21]  A. Bax,et al.  TALOS+: a hybrid method for predicting protein backbone torsion angles from NMR chemical shifts , 2009, Journal of biomolecular NMR.

[22]  G. Polis,et al.  PHENOLOGY AND LIFE HISTORY OF THE DESERT SPIDER, DIGUETIA MOJAVEA (ARANEAE, DIGUETIDAE) , 1999 .

[23]  G. Robertson,et al.  HERG, a human inward rectifier in the voltage-gated potassium channel family. , 1995, Science.

[24]  K. Dong.  Insect sodium channels and insecticide resistance , 2007, Invertebrate Neuroscience.

[25]  Vladimir Yarov-Yarovoy,et al.  Structure and Function of the Voltage Sensor of Sodium Channels Probed by a β-Scorpion Toxin* , 2006, Journal of Biological Chemistry.

[26]  R. Norton,et al.  The toxicogenomic multiverse: convergent recruitment of proteins into animal venoms. , 2009, Annual review of genomics and human genetics.

[27]  H. Zakon.  Adaptive evolution of voltage-gated sodium channels: The first 800 million years , 2012, Proceedings of the National Academy of Sciences.

[28]  W. Catterall,et al.  Voltage Sensor–Trapping Enhanced Activation of Sodium Channels by β-Scorpion Toxin Bound to the S3–S4 Loop in Domain II , 1998, Neuron.

[29]  G. King,et al.  Production of Recombinant Disulfide-Rich Venom Peptides for Structural and Functional Analysis via Expression in the Periplasm of E. coli , 2013, PloS one.

[30]  F. Bezanilla,et al.  Domain IV voltage-sensor movement is both sufficient and rate limiting for fast inactivation in sodium channels , 2013, The Journal of general physiology.

[31]  Francisco Bezanilla,et al.  A single charged voltage sensor is capable of gating the Shaker K+ channel , 2009, The Journal of general physiology.

[32]  Mehdi Mobli,et al.  Macromolecular NMR spectroscopy for the non‐spectroscopist , 2011, The FEBS journal.

[33]  K. Dong.  A single amino acid change in the para sodium channel protein is associated with knockdown-resistance (kdr) to pyrethroid insecticides in German cockroach. , 1997, Insect biochemistry and molecular biology.

[34]  Jack Snoeyink,et al.  Nucleic Acids Research Advance Access published April 22, 2007 MolProbity: all-atom contacts and structure validation for proteins and nucleic acids , 2007 .

[35]  P. Güntert.  Automated NMR structure calculation with CYANA. , 2004, Methods in molecular biology.

[36]  B. Hille.  Ionic channels of excitable membranes , 2001 .

[37]  F. Bezanilla,et al.  Tracking Voltage-dependent Conformational Changes in Skeletal Muscle Sodium Channel during Activation , 2002, The Journal of general physiology.

[38]  J. Haupt,et al.  Distinct primary structures of the major peptide toxins from the venom of the spider Macrothele gigas that bind to sites 3 and 4 in the sodium channel1 , 2003, FEBS letters.

[39]  Lei Fang,et al.  An improved strategy for high-level production of TEV protease in Escherichia coli and its purification and characterization. , 2007, Protein expression and purification.

[40]  G. King,et al.  Peptide toxins that selectively target insect NaV and CaV channels , 2008, Channels.

[41]  G. King,et al.  The insecticidal neurotoxin Aps III is an atypical knottin peptide that potently blocks insect voltage-gated sodium channels. , 2013, Biochemical pharmacology.

[42]  Francisco Bezanilla,et al.  Intermediate state trapping of a voltage sensor , 2012, The Journal of general physiology.

[43]  E. Zlotkin,et al.  A scorpion venom neurotoxin paralytic to insects that affects sodium current inactivation: purification, primary structure, and mode of action. , 1990, Biochemistry.

[44]  Mark W Maciejewski,et al.  An automated tool for maximum entropy reconstruction of biomolecular NMR spectra , 2007, Nature Methods.

[45]  D. Hanck,et al.  The Na channel voltage sensor associated with inactivation is localized to the external charged residues of domain IV, S4. , 1999, Biophysical journal.

[46]  H. Rochat,et al.  Scorpion α‐like toxins, toxic to both mammals and insects, differentially interact with receptor site 3 on voltage‐gated sodium channels in mammals and insects , 1999, The European journal of neuroscience.

[47]  E. Campbell,et al.  Atomic structure of a voltage-dependent K+ channel in a lipid membrane-like environment , 2007, Nature.

[48]  Mallur S. Madhusudhan,et al.  CLICK—topology-independent comparison of biomolecular 3D structures , 2011, Nucleic Acids Res..

[49]  G. King,et al.  Spider-Venom Peptides as Therapeutics , 2010, Toxins.

[50]  William A Catterall,et al.  Structure-Function Map of the Receptor Site for β-Scorpion Toxins in Domain II of Voltage-gated Sodium Channels* , 2011, The Journal of Biological Chemistry.

[51]  K. Krapcho,et al.  Characterization and cloning of insecticidal peptides from the primitive weaving spider Diguetia canities. , 1995, Insect biochemistry and molecular biology.

[52]  R. MacKinnon,et al.  Hanatoxin Modifies the Gating of a Voltage-Dependent K+ Channel through Multiple Binding Sites , 1997, Neuron.

[53]  G. King,et al.  Spider-venom peptides that target voltage-gated sodium channels: pharmacological tools and potential therapeutic leads. , 2012, Toxicon : official journal of the International Society on Toxinology.

[54]  A. A. Alabi,et al.  Portability of paddle motif function and pharmacology in voltage sensors , 2007, Nature.

[55]  J. Ruppersberg.  Ion Channels in Excitable Membranes , 1996 .

[56]  W. Eberhard.  Attack behavior of diguetid spiders and the origin of prey wrapping in spiders , 1967 .

[57]  P. Deák,et al.  Cloning and functional analysis of tipE, a novel membrane protein that enhances drosophila para sodium channel function , 1995, Cell.

[58]  Mirela Milescu,et al.  Interactions between lipids and voltage sensor paddles detected with tarantula toxins , 2009, Nature Structural &Molecular Biology.

[59]  Werner Treptow,et al.  Intermediate states of the Kv1.2 voltage sensor from atomistic molecular dynamics simulations , 2011, Proceedings of the National Academy of Sciences.

[60]  Wolfgang Bermel,et al.  A non-uniformly sampled 4D HCC(CO)NH-TOCSY experiment processed using maximum entropy for rapid protein sidechain assignment. , 2010, Journal of magnetic resonance.

[61]  A. L. Goldin,et al.  Alternative Splicing of an Insect Sodium Channel Gene Generates Pharmacologically Distinct Sodium Channels , 2002, The Journal of Neuroscience.

[62]  W. Catterall,et al.  Voltage-gated ion channels and gating modifier toxins. , 2007, Toxicon : official journal of the International Society on Toxinology.

[63]  K. Dong,et al.  Intron Retention in mRNA Encoding Ancillary Subunit of Insect Voltage-Gated Sodium Channel Modulates Channel Expression, Gating Regulation and Drug Sensitivity , 2013, PloS one.

[64]  Liisa Holm,et al.  Dali server: conservation mapping in 3D , 2010, Nucleic Acids Res..

[65]  J. Bloomquist,et al.  Mode of action of an insecticidal peptide toxin from the venom of a weaving spider (Diguetia canities). , 1996, Toxicon : official journal of the International Society on Toxinology.

[66]  G. King,et al.  NMR methods for determining disulfide-bond connectivities. , 2010, Toxicon : official journal of the International Society on Toxinology.

[67]  W. Catterall,et al.  THE CRYSTAL STRUCTURE OF A VOLTAGE-GATED SODIUM CHANNEL , 2011, Nature.

[68]  A. L. Goldin,et al.  Use-dependent potentiation of the Nav1.6 sodium channel. , 2004, Biophysical journal.

[69]  Jianhua He,et al.  Crystal structure of an orthologue of the NaChBac voltage-gated sodium channel , 2012, Nature.

[70]  Roderick MacKinnon,et al.  Contribution of the S4 Segment to Gating Charge in the Shaker K+ Channel , 1996, Neuron.

[71]  K. Elmslie,et al.  Slowed N-type calcium channel (CaV2.2) deactivation by the cyclin-dependent kinase inhibitor roscovitine. , 2005, Biophysical journal.

[72]  K. Swartz,et al.  Targeting voltage sensors in sodium channels with spider toxins. , 2010, Trends in pharmacological sciences.