Analysis of Voltage-gated Ion Channels in Antarctic Fish

该项目研究生活在南极洲的鱼的神经和肌肉的蛋白质在寒冷中如何发挥作用，这应该会提供关于这些相同蛋白质在包括人类在内的所有动物中的功能的信息。这些被称为离子通道的蛋白质，打开和关闭，允许离子(带电荷的原子或分子)流入或流出细胞，从而引起神经和肌肉的电活动。影响这一过程的突变是许多人类疾病的基础，如癫痫、心律失常和肌肉瘫痪。因此，了解蛋白质的哪些部分控制这些转变是很重要的。通道打开和关闭的速度取决于温度。人体离子通道在寒冷时转换缓慢，这就是为什么我们在寒冷中变得麻木。然而，被称为冰鱼的南极鱼在极大地限制人类离子通道活动的冰点温度下活动。研究人员有证据表明，银鱼离子通道的特定变化使它们的通道在寒冷中以不同的方式运行，他们将使用基因发现和生物物理方法来测试这些变化如何改变不同温度下冰鱼蛋白质的转变。该项目还将推动NSF培养新一代科学家和向公众提供科学发现的目标。基因发现分析将由本科生完成，其中包括一所为少数族裔服务的大学的本科生，研究人员将开发一门新课程，该课程也将服务于该机构和其他地方的学生。此外，调查人员将与公众以及有特殊需要的团体一起参加教育外展活动。诺尼奥鱼是南极洲最成功的脊椎动物之一。没有生物能够适应它们居住的冰冻的水，这个项目将研究它们的电压门控离子通道(VGIC)如何在寒冷中发挥作用。离子通道的分子运动因寒冷而严重受损，但Notothens的功能在温度上会使“冷血”温带动物的神经和肌肉瘫痪。令人惊讶的是，还没有对当时的VGIC进行生物物理或分子研究。研究人员有初步数据表明，VGICs中的氨基酸替换发生在从果蝇到人类的进化保守的位置。其中一些已知影响通道功能，而另一些在通道转换中的作用尚不清楚。对它们的研究结果将提供同样适用于非自然、甚至是人类的VGIC的新信息，并为VGIC如何适应寒冷提供洞察力。该项目将使用电压钳制技术对NOTO VGIC进行生物物理表征，并从两个温带FISH将它们在不同温度范围内的性能与同一通道进行比较。独特的NOTO氨基酸替换的作用将以突变为特征。一个具体的目标将是一个项目，在该项目中，本科生将挖掘NOTO序列数据库，以确定VGIC中可能有助于适应寒冷的其他潜在氨基酸替代。

This project studies how the proteins of the nerves and muscles of fish that live in Antarctica function in the cold, which should provide information on the function of these same proteins in all animals, including humans. These proteins, called ion channels, open and close to allow ions (atoms or molecules with electrical charge) to flow into or out of cells which causes the electrical activity of nerves and muscles. Mutations that influence this process are the basis of numerous human disorders such as epilepsy, heart arrhythmias, and muscle paralysis. Thus, it is important to understand what parts of the proteins govern these transitions. The speed with which channels open and close depends on temperature. Human ion channels transition slowly when we are cold, which is why we become numb in the cold. Yet Antarctic fish, called icefish, are active at freezing temperatures that drastically limit the activity of human ion channels. The investigators have evidence that specific alterations in the icefishs' ion channels allow their channels to operate differently in the cold and they will use gene discovery and biophysical methods to test how these changes alter the transitions of icefish proteins at different temperatures. The project will also further the NSF goals of training new generations of scientists and of making scientific discoveries available to the general public. The gene discovery analysis will be done by undergraduate students including those from a minority-serving university and the investigators will develop a new course which will also serve students at that institution and elsewhere. In addition, the investigators will participate in educational outreach events with the general public as well as with groups with special needs.Notothenioid fishes are one of the most successful groups of vertebrates in Antarctica. Notothens have adaptations to the freezing water they inhabit and this project will study how their voltage-gated ion channels (VGICs) function in the cold. The molecular movements of ion channels are severely impaired by cold, yet notothens function at temperatures that would paralyze the nerves and muscles of "cold-blooded" temperate zone animals. Surprisingly, no biophysical or molecular investigations have been conducted on notothen VGICs. The investigators have preliminary data that amino acid substitutions occur at sites in VGICs that are evolutionarily conserved from fruit flies to humans. Some of these sites are known to impact channel function and the role of others in channel transitioning are unknown. The results from studying them will provide novel information also applicable to non-notothen, perhaps even human, VGICs as well as providing insights into how VGICs adapt to the cold. The project will biophysically characterize notothen VGICs using voltage-clamp techniques will and compare their properties over a range of temperatures to the same channel from two temperate zone fish. The role of unique notothen amino acid substitutions will be characterized by mutagenesis. One specific aim will be a project in which undergraduates mine notothen sequence databases to identify other potential amino acid substitutions in VGICs that might facilitate adaptation to the cold.