The price of success: biophysical costs of tetrodotoxin resistance in voltage-gated sodium channels

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Garter snakes (Thamnophis sirtalis) are sympatric with their prey. Tetrodotoxic newts (Taricha granulosa) have evolved tetrodotoxin resistance that allows the snakes to ingest toxic newts. Allopatric snakes are tetrodotoxin-sensitive. The level of resistance in snakes spans a wide range and roughly correlates with the toxicity of the newts. This system is thus a co-evolutionary arms race. Voltage-gated sodium channels are responsible for the rising phase of the action potential in electrically excitable cells. Our previous research has identified up to four amino acid substitutions in the skeletal muscle voltage-gated sodium channel. These substitutions account for toxin resistance. Furthermore, the number of substitutions correlates with the level of resistance. The substituted residues are located within the pore-forming region of the sodium channel, which is an otherwise highly conserved region because of its critical function in ion selectivity and permeation. Although we have characterized the toxin sensitivity of the skeletal muscle sodium channel from four different populations of snakes (apparently representing nearly the full range of toxin resistance), we have yet to characterize the biophysical properties, including gating and selectivity, of these sodium channels. These properties may be particularly relevant to this system, since it has been observed that the maximum crawl speed of resistant snakes is slower than in non-resistant animals. To fully understand the implications of toxin resistance, and to elucidate the costs of the amino acid substitutions to sodium channel function, we now seek to study the biophysical properties of sodium channels from the four populations of snakes. Sodium channels will be expressed in Xenopus oocytes and their biophysical properties will be assessed using patch clamp techniques. We will study the voltage dependence and kinetics of channel gating as well as the selectivity and single channel conductance of snake skeletal muscle sodium channels. This study will allow us to determine whether there are biophysical costs to toxin resistance and will lead to a greater understanding of the structure/function relationships that control sodium channels.