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

离子通道是一种特殊的蛋白质，存在于细胞膜上，在所有生命形式中塑造神经系统的电活动。编码离子通道蛋白的基因中自然发生的变异（突变）可以决定整个神经系统的电特性。为了更好地理解离子通道的序列、结构和功能之间的关系，研究人员将利用仅在非洲弱电鱼类中发现的钾通道基因突变。他们假设这些突变赋予了异常快速的分子运动，从而产生了快速的电活动，使这些鱼类能够产生用于通信和导航的快速电脉冲。这项拨款的第一个目标是对来自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。第二个目标将是在体外表达这些基因，以研究突变赋予的物理特性。这项工作很重要，因为它使我们更深入地了解遗传变化在决定所有类型细胞的电特性方面所起的作用，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和实验室经验培训本科生，包括那些来自科学领域代表性不足的群体的本科生。研究人员将研究非洲电鱼的电器官（肌肉的衍生物）中钾通道（Kv）的序列进化与生物物理特性之间的关系。大多数电器官放电（eod）用于通信和导航，并且在这一群体中非常短暂（500微秒），然而少数物种二级进化出长时间放电。一个Kv通道（kcna7a）在电器官中大量表达，初步数据表明，该蛋白高度保守区域的序列进化和氨基酸取代率很高，可能赋予其独特的生物物理特性。在第一个目标中，研究人员将对10种非洲电鱼的电器官和肌肉组织进行RNAseq，并检查kcna7a序列进化与EOD表型进化的关系。在第二个目标中，研究人员将在目标1的指导下对kcna7a通道基因进行定点诱变，在青蛙卵母细胞中表达诱变通道，并进行生理记录以确定特定氨基酸赋予的生物物理特性。这项工作将深入了解参与物种形成的通信信号快速进化的遗传基础；研究一类在形成神经活动中普遍重要的医学相关离子通道中的新型氨基酸取代；可能为组织工程中形成电活动的超快速动力学通道提供资源，并为实验室研究电器官发育和进化的其他方面提供转录组学资源。

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.