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

吊带蛇（Thamnophis sirtalis）与它们的猎物是同乡的。河豚毒蝾螈（Taricha granulosa）已经进化出了河豚毒素抗性，使蛇能够摄入有毒的蝾螈。异域蛇对河豚毒素敏感。蛇的抗药水平范围很广，大致与蝾螈的毒性有关。因此，这个系统是一场共同进化的军备竞赛。电压门控钠通道负责电兴奋性细胞的动作电位上升相。我们之前的研究已经确定了骨骼肌电压门控钠通道中多达四个氨基酸的取代。这些替代解释了毒素抗性。此外，替换的数量与抗性水平相关。取代残基位于钠通道的成孔区域内，由于其在离子选择性和渗透方面的关键作用，该区域是一个高度保守的区域。虽然我们已经描述了四种不同蛇类的骨骼肌钠通道的毒素敏感性（显然代表了几乎所有的毒素抗性），但我们还没有描述这些钠通道的生物物理特性，包括门控和选择性。这些特性可能与这个系统特别相关，因为已经观察到，抗性蛇的最大爬行速度比非抗性动物慢。为了充分了解毒素抗性的含义，并阐明氨基酸取代对钠通道功能的影响，我们现在试图研究四种蛇的钠通道的生物物理特性。钠离子通道将在非洲爪蟾卵母细胞中表达，并利用膜片钳技术评估其生物物理特性。我们将研究通道门控的电压依赖性和动力学，以及蛇骨骼肌钠通道的选择性和单通道电导。这项研究将使我们能够确定毒素抗性是否有生物物理代价，并将使我们更好地了解控制钠通道的结构/功能关系。

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.