Collaborative Research: Analysis of a Rapidly Evolving Potassium Channel in an Electric Fish

合作研究：分析电鱼中快速进化的钾通道

• 批准号: 1557837
• 负责人: Amanda Masino
• 金额: $ 10万
• 依托单位: Huston-Tillotson University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-07-01 至 2020-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1557837&HistoricalAwards=false
• 关键词: Collaborative; Research; Analysis; Rapidly; Evolving

Ion channels, specialized proteins that reside in the cell membrane, shape the electrical activity of nervous systems in all forms of life. Naturally occurring variation (mutations) in genes that encode ion channel proteins can determine electrical properties throughout the nervous system. To better understand the relationship between sequence, structure and function of ion channels, the investigators will use mutations discovered in a potassium channel gene found only among the weakly electric fishes of Africa. They hypothesize that these mutations confer extraordinarily rapid molecular movements, and thus rapid electrical activity, enabling these fishes to produce rapid pulses of electricity used in communication and navigation. The first aim of this grant will be to sequence this gene from a variety of African electric fishes to determine the evolutionary origin of this mutation. The second aim will be to express these genes in-vitro to investigate the physical properties that the mutation confers. This work is important because it gives us greater insight into the role that genetic changes play in determining electrical properties of all types of cells, including heritable diseases of the nervous system (channelopathies), as well as adaptive differences that may shape the nervous system in the evolution of new behaviors. As part of their work, the investigators will train undergraduates, including those from underrepresented groups in science, through coursework and laboratory experiences in molecular evolution, physiology and genomics.Investigators will investigate the relationship between sequence evolution and biophysical properties of a potassium channel (Kv) exclusively expressed in the electric organ, a derivative of muscle, in African electric fish. Most electric organ discharges (EODs), are used for communication and navigation, and are extraordinarily brief (500 microseconds) within this group, however a few species have secondarily evolved long duration discharges. One Kv channel (kcna7a) is abundantly expressed in the electric organ, and preliminary data suggests high rates of sequence evolution and amino acid substitutions in otherwise highly conserved regions of this protein, likely conferring unique biophysical properties. In the first aim investigators will perform RNAseq on electric organ and muscle tissues from 10 species of African electric fish strategically chosen for their phylogenetic relationships and waveform duration, and examine kcna7a sequence evolution as it relates to EOD phenotypic evolution. In the second aim, investigators will perform site-directed mutagenesis on kcna7a channel genes, guided by discoveries in aim 1, express mutagenized channels in frog oocytes, and perform physiological recordings to determine biophysical properties conferred by specific amino acids. This work will give insights into the genetic basis of rapid evolution of a communication signal involved in speciation; investigate novel amino acid substitutions in a class of medically-relevant ion channels that are universally important in shaping neural activity; potentially provide resources for making channels with hyper-fast kinetics for shaping electrical activity in tissue engineering and provide transcriptomic resources for laboratories studying other aspects of electric organ development and evolution.

离子通道是一种驻留在细胞膜上的特殊蛋白质，它塑造了所有形式生命中神经系统的电活动。编码离子通道蛋白的基因自然发生的变异(突变)可以决定整个神经系统的电特性。为了更好地了解离子通道的序列、结构和功能之间的关系，研究人员将使用在钾通道基因中发现的突变，这种突变只在非洲弱电鱼类中发现。他们假设，这些突变赋予了非常快速的分子运动，因此快速的电活动，使这些鱼能够产生用于通信和导航的快速电脉冲。这笔赠款的第一个目标将是对各种非洲电鱼的这种基因进行测序，以确定这种突变的进化起源。第二个目标是在体外表达这些基因，以研究突变带来的物理特性。这项工作很重要，因为它让我们更深入地了解了基因变化在决定所有类型细胞的电特性中所起的作用，包括神经系统的遗传性疾病(通道病)，以及可能在新行为的进化中塑造神经系统的适应性差异。作为他们工作的一部分，研究人员将通过分子进化、生理学和基因组学方面的课程作业和实验室经验对本科生进行培训，包括那些在科学领域未被充分代表的群体的本科生。调查人员将调查非洲电鱼中唯一表达的钾通道(Kv)的序列进化与生物物理特性之间的关系。大多数电子器官放电(EOD)用于通信和导航，并且在这一组中非常短暂(500微秒)，然而少数物种已经次级进化出长时间的放电。一个Kv通道(Kcna 7a)在电子器官中大量表达，初步数据表明该蛋白在其他高度保守的区域有很高的序列进化和氨基酸替换速率，可能赋予了独特的生物物理特性。在第一个目标中，研究人员将对根据系统发育关系和波形持续时间战略选择的10种非洲电鱼的电子器官和肌肉组织进行RNAseq，并检查kcna7a序列进化与EOD表型进化有关。在第二个目标中，研究人员将在目标1的发现指导下，对kcna 7a通道基因进行定点突变，在青蛙卵母细胞中表达突变的通道，并进行生理记录，以确定特定氨基酸赋予的生物物理性质。这项工作将深入了解与物种形成有关的通讯信号快速进化的遗传基础；研究一类与医学相关的离子通道中的新氨基酸替换，这些离子通道对塑造神经活动具有普遍重要的作用；可能为制造具有超快动力学的通道来塑造组织工程中的电活动提供资源，并为实验室研究电子器官发育和进化的其他方面提供转录资源。