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

成功的代价：电压门控钠通道河豚毒素抗性的生物物理成本

• 批准号: 342155-2007
• 负责人: Ruben, Peter
• 金额: $ 2.55万
• 依托单位: Simon Fraser University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2011
• 资助国家: 加拿大
• 起止时间: 2011-01-01 至 2012-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=482439
• 关键词: price; success; biophysical; costs; tetrodotoxin

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.

加特蛇(Thamnopits Sirtalis)与猎物共处一地。河豚(Taricha Graosa)已经进化出河豚毒素抗性，使蛇能够摄取有毒的蝾螈。异地蛇对河豚毒素敏感。蛇的抵抗力水平范围很广，并与蝾螈的毒性大致相关。因此，这一体系是一场共同进化的军备竞赛。电压门控钠通道负责电兴奋细胞动作电位的上升阶段。我们之前的研究已经在骨骼肌电压门控钠通道中发现了多达四个氨基酸替代。这些替换是产生毒素抗性的原因。此外，替换的数量与抗药性水平相关。被取代的残基位于钠通道的成孔区，这是一个高度保守的区域，因为它在离子选择性和渗透方面具有关键作用。虽然我们已经表征了来自四个不同种群的蛇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)，但我们还没有表征这些钠通道的生物物理性质，包括门控和选择性。这些特性可能与这个系统特别相关，因为已经观察到抗药性蛇的最大爬行速度比非抗药性动物慢。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性质。钠通道将在非洲爪哇卵母细胞中表达，并将使用膜片钳技术评估其生物物理特性。我们将研究电压依赖性和通道门控的动力学，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是否存在毒素抗性的生物物理成本，并将有助于更好地理解控制钠通道的结构/功能关系。