Interactions of insecticidal spider peptide neurotoxins with insect voltage- and neurotransmitter-gated ion channels
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Two families of peptide neurotoxins that target insect large-conductance calcium-activated potassium channels (BKCa) have been isolated from the venom of two unrelated spiders. The ƙ-TRTX-Ec2 toxins are a family of three homologous peptides isolated from the African tarantula, Eucratoscelus longiceps and ƙ-HXTX-Hv1c is the prototypic member of a family of insect-selective neurotoxins isolated from the venom of the Blue Mountains funnel-web spider, Hadronyche versuta. This thesis describes the characterisation of these insecticidal toxins using voltage-clamp and current-clamp analysis of cockroach dorsal unpaired neurons utilising the whole-cell patch-clamp technique. The ability of these toxins to modulate the gating and kinetics of both voltage- and neurotransmitter-gated ion channels were assessed. Insect bioassays were also utilised to validate the insecticidal activities of various toxins that target KV channel subtypes in house crickets. The ƙ-TRTX-Ec2 family of toxins were found to be high affinity blockers of the insect BKCa channel while failing to modify voltage-gated sodium (NaV) and calcium (CaV) channels. ƙ-TRTX-Ec2a, -Ec2b and -Ec2c block cockroach BKCa channels with IC50 values of 3.7, 25.3 and 24.6 nM, respectively. Additionally, ƙ-TRTX-Ec2a was found to inhibit delayed-rectifier KV channel currents (IK(DR)), but only at significantly higher concentrations. ƙ-TRTX-Ec2 toxins induced voltage-independent channel block and are thus proposed to interact with the turret and/or loop region of the external vestibule of the insect BKCa channel. ƙ-HXTX-Hv1c has also been characterised to block the insect BKCa channel, while failing to modulate insect NaV and CaV channels. The unique insect-selective action of ƙ-HXTX-Hv1c involves a rare vicinal disulphide ring (Cys13-Cys14) that has been determined to act as part of the bioactive surface (pharmacophore) interacting with the molecular recognition site on the insect BKCa channel. However, despite the high affinity and selectivity for the BKCa channel it was discovered that the BKCa channel is unlikely to be the lethal target of ƙ-HXTX-Hv1c. Acute toxicity tests of classical non-phylum selective BKCa blockers such as paxilline, charybdotoxin and iberiotoxin did not induce acute toxicity in insects. Furthermore, while ƙ-HXTX-Hv1c was found to prolong action potential repolarisation, increase spontaneous firing frequency and reduce spike afterhyperpolarisation, these results were markedly reduced in the presence of the BKCa channel blocker iberiotoxin. Subsequent testing of cockroach KV channel currents revealed that ƙ-HXTX-Hv1c failed to modify sodium-activated or delayed-rectifier KV channel currents, but 1 µM ƙ-HXTX-Hv1c did produce a 29% block of ‘A-type’ fast-transient KV channel currents (IK(A)). This suggests that ƙ-HXTX-Hv1c additionally targets insect KV1- or KV4-like channel subtypes. The lethal insecticidal action of 4-AP in crickets further supports an action of ƙ-HXTX-Hv1c to block IK(A). The results of co-application experiments revealed that ƙ-HXTX-Hv1c blocks the same channel as the non-phylum selective vertebrate KV4 channel toxin, ƙ-sparatoxin-Hv1b. However, it was found that ƙ-sparatoxin-Hv1b, either alone or in combination with iberiotoxin, was not insecticidal and thus the KV4 and BKCa channels are unlikely to be the lethal targets of ƙ-HXTX-Hv1c. To determine if the lethal target was a neurotransmitter-gated ion channel, the effects of ƙ-HXTX-Hv1c were investigated on chloride-gated GABAA (GABA-Cl) and glutamate (Glu-Cl) channel currents and nAChR channel currents. It was revealed that 1 µM ƙ-HXTX-Hv1c failed to modify GABAA channel currents while causing only a moderate 21% increase in Glu-Cl channel currents. Alternately, it was found that ƙ-HXTX-Hv1c caused a concentration-dependent (EC50 183 nM) slowing of nicotinic acetylcholine receptor (nAChR) channel current decay and reversed channel desensitisation. In addition, ƙ-HXTX-Hv1c moderately increased nAChR sensitivity to nicotine. These findings are consistent with a positive allosteric modulation of insect nAChRs to slow receptor desensitisation. The nAChR is a validated insecticidal target for various agrochemical insecticides, including the allosteric modulator spinosyn A. Therefore it is believed that the lethal target of ƙ-HXTX-Hv1c is the insect nAChR, whose modulation would lead to an increase in neurotransmission consistent with the excitotoxic phenotype of the toxin. This action is possibly augmented by additional actions on BKCa and KV4 like channels to increase neuronal excitability.
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