Coevolution of tetrodotoxin and voltage-gated sodium channels

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The long-term goal of this research program is to understand the co-evolution of toxins and their targets. The model we use to understand the basis of toxin/target co-evolution is the interaction between tetrodotoxin (TTX) and the voltage-gated sodium channel (NaV). The interaction between these two entities is highly specific; NaV is the only known substrate for TTX. Although the physiological and biophysical consequences of TTX binding to NaVs are well documented, and although the NaV structures associated with TTX binding are understood, technical limitations have restricted the ability to manipulate the structure of TTX. We therefore have only a limited knowledge about the nature of the TTX/NaV interaction from the toxin perspective. Our published, NSERC-funded research shows that NaVs have independently evolved different structural mechanisms to reduce their sensitivity to TTX, although these adaptations have physiological consequences to the organisms in which they have evolved. This suggests a dynamic nature to the TTX/NaV interaction that may be reflected in structural changes on both sides of the relationship. Therefore, the aim of this phase of our research program is to focus on the structure of TTX. We will produce structural variants of TTX and test these variants for their ability to block the sodium current in different NaV isoforms, and to alter the gating current in these isoforms. To measure ion and gating currents, we will express sodium channels in Xenopus oocytes and record currents using the cut-open oocyte technique. The specific aims of the present proposal are: 1) produce TTX variants to test the role of each OH group, the guanidine segment, and urea groups in TTX; 2) study the concentration-dependence of ion current block of TTX variants in NaV1.4, NaV1.5, and tsNaV1.4; 3) study the concentration-dependence of shifts in the voltage-dependence of gating currents produced by the TTX variants in NaV1.4, NaV1.5, and tsNaV1.4. The significance of this phase of our program is that it will reveal the structural requirements of TTX for its effects on NaV and improve our understanding of how the structure of TTX may have co-evolved with sodium channels.