The Interaction Of Receptor Site 3 And 4 Toxins With Insect Sodium Channels

Part one:The study of kinetics property of BgNav sodium channelsBased on the sequence of BgNavl-1a sodium channel which is a variant of BgNav,an insect sodium channel extracted from Blattella germanica.With the technique of site-directed mutagenesis,charge reversed amino acid residues in the voltage-sensing module of domain Ⅲ got us five mutants E1268K (IIIS1-E1K) E1273K (IIIS1-E2K),D1308K (IIIS2-D1K),E1318K (IIIS2-E2K) and R4E.Expressed them in Xenopus Oocytes.The study of voltage-dependence of these five mutants showed no differences when compared with control-BgNavl-1a sodium channel.Both the voltage-dependence of activation and inactivation of these five mutants shifted to the same direction with no big amplitude.Part two:The interaction of β scorpion toxin Lqh-dprIT3 with BgNavl-la and its mutantsScorpion_-toxins bind to the extracellular regions of the voltage-sensing module of domain Ⅱ and to the pore module of domain Ⅲ in voltage-gated sodium channels and enhance channel activation by trapping and stabilizing the voltage sensor of domain Ⅱ in its activated state. We investigated the interaction of a highly potent insect-selective scorpion depressant β-toxin,Lqh-dprIT3, from Leiurus quinquestriatus hebraeus with insect sodium channels BgNavl-1a and its mutants. D1308K (IIIS2-D1K),E1318K (ⅢS2-E2K) increased the sensitivity of the channels to Lqh-dprIT3. These findings further proved the involvement of the domain Ⅲ voltage-sensing module in the action of scorpion P-toxins.Part three:The interaction of receptor site 3 toxins with BgNavl-la and its mutantsThe a-scorpions toxins bind to the resting state of Na+channels and inhibit fast inactivation by interaction with a receptor site formed by domains I and IV.As one short-chain toxin which extracted from Anemonia viridis,Av3 bing to the receptor site 3 to inhibit fast inactivation of sodium channels similar to a-scorpions toxins.. We investigated the interaction of a a-scorpions toxin LqhαIT and Av3 with insect sodium channels BgNavl-1a and its mutants. D1308K (IIIS2-D1K),E1318K (IIIS2-E2K) increased the sensitivity of the channels to LqhαIT and Av3.Charge reversed the forth positive residue in IIIS4 also increased the sensitivity of the channels to LqhαIT and Av3.The noval findings showed the involvement of the domain Ⅲ voltage-sensing module in the action of receptor site 3 toxins and increase S4 trapping via allosteric mechanisms, suggesting coupling between the voltage sensors in neighboring domains during channel activation.