The Molecular Mechanism Of Av3 Sea Anemone Toxin Selective Action On Insects

BackgroundElucidation of the way toxic ligands interact with voltage-gated sodium channels(Navs)is instrumental in the study of channel structure-activity relationship,and may also be useful for drug design.Voltage-gated sodium channels is targeted by a variety of structurally distinct polypeptide toxins and insecticides from scorpions,spiders and sea anemones.Three groups of sea anemone toxins(type ?,type ? and type ?)have been shown to inhibit Nav inactivation,but only one point of putative interaction between a type ? sea anemone toxin and a Nav has thus far been proposed(Lys37 in anthopleurin B with Asp 1612 at DIV/S3-S4 of hNav1.5).Among these groups,the type? toxin Av3(27 amino acids)from Anemonia viridis(previously Anemonia sulcata)exhibits a unique structure of only turn-based secondary-structure elements reticulated by three disulfide bonds and lacking ?-helices or ?-strands which make the bioactive surface of Av3 a intriguing issue.As one of the Site-3 neurotoxin,Av3 has been shown to be active only on insect sodium channels,but not on mammalian counterparts.More detailed description of receptor site-3 for scorpion ?-toxins was reported more recently and included linkers S1-S2 and S3-S4 at the voltage-sensing module of DIV and the adjacent external linker S5-S6 at the pore module of DI.We presumed that the binding site of Av3 with insect sodium channel involved in the linker mentioned above.In this study,bioactive assay and electrophysiological technology are utilized to investigate the bioactive surface of Av3 in vivo and vitro;and we will identify unique sequences in the insect sodium channel that interact with Av3 by systematically replacing the extracellular loop sequences of Av3-sensitive BgNav1-1a channel from Blattella germanica with corresponding loop sequences of Av3-insensitive rat brain rNav1.2a channel and vice versa.Combined with electrophysiological technology,the analysis results will reveal the loops even the residues which are critical for Av3 selective action on insect sodium channel.Accomplishment of the proposed work will uncover the selectivity mechanism of Av3 on insect sodium channels.Such knowledges would be highly valuable for future design of new selective anti-insect compounds.ObjectiveDouble electrodes voltage clamp building combined with chemical synthesis,bioactive assay,chimera building and site-directed mutagenesis are utilized to investigate the molecular mechanism of Av3 selective action on insect sodium channel BgNav1-1aMethods1)Chemical synthesized Av3 and Av3-based mutants were used in the following bioactive assay and electrophysiology experiments to investigate the bioactive surface of Av3 and binding site on insect sodium channel BgN avl-la.And RP-HPLC and ESI-MS were used to identify the purity and molecular weight of polypeptides.2)In bioactive assays,the acute toxicity of Av3 and Av3-based mutants were tested in German cockroaches and American cockroaches.Toxins were dissolved in insect saline and injected into the ventro-lateral thoracic region of adult American cockroaches(Periplaneta amricana)and adult German cockroaches(Blattella germanica).The positive results for knockdown were scored when immobilization and contraction were observed after 20-30 min.The lethal activity was then determined over a 24h period.Five concentrations of each toxin were injected into Blattella germanica(nine adults in each group)and Periplaneta Americana(ten adults in each group)in two independent experiments.Dose-response curves to determine LD50 and KD50 values were fitted using the logistic equation.3)First,we will identify unique sequences in the insect sodium channel that interact with the Av3 toxin by systematically replacing the extracellular loop sequences of the Av3-sensitive BgNav1-1a channel from Blattella germanica with corresponding loop sequences of the Av3-insensitive rat brain rNav1.2a channel.Next,we will identify specific amino acid residues in the relevant loop sequences that are critical for BgNavl-1 a interaction with Av3.4)The PGH19 vectors bearing the genes encoding for the insect sodium channels BgNav,1-1a and rat brain sodium channel rNav1.2a were a g ift from Dr Ke Dong(Department of Insect Toxicology And Neurobiology,Michigan State University,East Lansing,MI,U.S.A.).cRNAs encoding BgNav,1-1a,rNav1.2a and auxiliary subunit TipE were linearized and transcribed by NoTI and T7 RNA-polymerase and the mMESSAGE mMACHINE(?)system(Ambion).5)Double electrode voltage clamp technology was used in two parts:one is the action of Av3 and Av3-based mutants on insect sodium channel BgNav1-1a;The other is action of Av3 on chimera and mutants which were made based on BgNav1-1a and rNav1.2a.The mass ratios for ?:? subunits injected were 1:1 for insect channels.Av3 ability to affect the channel fast-inactivation process was examined while the peak current was around 2 ?A.Results1)The bioactivity of chemical synthesized Av3 and Av3-based mutants were confirmed by bioactive assay and electrophysiological assay.2)Three mutants Y7A,W8A and Y18A reduced the sensitivity of toxin to adult German Cockroaches,and the KD50 of the three mutants increased more than 1O-fold compaired with Av3-WT which made us presumed that the three aromatic residues Tyr7,Trp8 and Tyr18 involved in the bioactive surface of Av3.3)In the test of Av3-WT action on adult American Cockroaches,we found unexpectly that the low sensitivity of adult American Cockroaches to Av3-WT,the KD50 for American Cockroaches increased more than 50-fold than that of German Cockroaches.4)Electrophysiological assay show us the different degree of inhibition of channel inactivation by Av3 and Av3-based mutants.Y7A,W8A and Y18A significantly decreased the effect of toxin in inactivation inhibition.I20/Ipeak of Y7A,W8 A and Y18A was 0.119,0.234 and 0.085,respectively.Another intriguing issue is S23A and P25A increased the toxin action on inactivation inhibition,I2o/IPeak of S23A and P25A were 0.883 and 1,while that of Av3-WT was 0.68.5)In the electrophysiology assay,the chimera bearing DI/SS2-S6 of the mammalian channel lost sensitivity to Av3.This result has sorted out DI/SS2-S6 as the external linker involved in determining Av3 specificity for the BgNav1-1a channel.6)Inbolved of DI/SS2-S6 in Av3 specificity prompted us to analyze which residues in DI/SS2-S6 affect the Av3 specificity.DI/SS2-S6 of BgNav1-1a differs only slightly from its rNav1.2a equivalent.We examined the role of five residues at this linker.Va1395,Ser398,Pro402,Trp403 and His404 in BgNav1-1a versus their Lys,Thr and Tyr equivalents in rNav1.2a.Substitution V396T,S399A,P402K and W403T had no effect,whereas His404Y completely abolished the toxin effect.And the minute effect of substitution H404A and strong effect of substitution H404F on Av3 action.Conclusions1)The biological activity of chemical synthesized polypeptides toxin Av3 and Av3-based mutants were identified by bioactive assay and electrophysiological assay.2)Three aromatic residues Tyr7,Trp8 and Tyr18 involved in the constitution of bioactive surface of Av3,affect the Av3 specificity;Side chain of Ser23 and Pro25 in Av3 restricted the effect of Av3 specificity which suggest the improvement in the insecticidal potency of naturally occurring substances is feasible.3)Selective action of Av3 between German Cockroaches and American Cockroaches maybe imply the different subset of Nav channel splice variants exist in German Cockroaches and American Cockroaches or the presence of endogenous pharmacologically active auxiliary subunits in German Cockroaches.4)DI/SS2-S6 of BgNav1-1a as the external linker involved in determining Av3 specificity for the German Cockroaches channel,furthermore His404 in DI/SS2-S6 is critical for Av3 selective action on BgNav1-1a.And the aromatic side chain at position 404 in the channel clearly hinders Aav3 binding and action.