RUI: Functional characterization of Na+/H+ exchangers in marine and freshwater fishes

鱼鳃在陆生动物中起着肺、肝和肾的作用。这个器官提供气体交换、代谢废物排泄、盐平衡和酸碱调节的场所。鳃细胞中的一种特殊蛋白质被认为可以通过细胞膜将钠交换为氢（Na+/H+交换；NHE），并与其他蛋白质转运蛋白结合，使鱼能够调节血液和组织中的酸（pH）水平。NHE在许多哺乳动物和非哺乳动物系统中都有描述。这些蛋白被认为具有多种功能，包括细胞体积调节、pH平衡和Na+摄取。在哺乳动物中已经发现了几种不同类型的NHE，每一种都由不同的基因编码。位于顶端的NHE2和NHE3被认为是转运上皮组织（如肾和肠）的特异性蛋白。在本研究中，研究人员重点研究了NHE3反转运蛋白在鱼鳃中的功能，以及该蛋白在海洋和淡水鱼的酸碱和离子调节中的作用。利用分子生物学方法，研究人员克隆了海洋长角鱼（Myoxocephalus octodecimspinosus）鳃中的全长NHE3转录本。雕塑NHE3与哺乳动物NHE3同源性高。该序列还包括编码区内可变的小卫星重复序列。这个插入的功能（或效果）是未知的。NHE3 mRNA的Northern印迹分析表明，在酸中毒后0.5-2小时，NHE3 mRNA的转录量增加，转录量为4.5 kb。用NHE3反义探针对鳃切片进行原位杂交，鉴定出鳃细胞，这些细胞被认为也表达NHE2和Na+/K+- atp酶。利用鱼类特异性抗体对适应淡水的异克莱底鱼（Fundulus heteroclitus）和鲫鱼（Carassius Carassius）的特定鳃细胞（假定是氯细胞）顶端表面的蛋白质进行免疫检测。研究人员提出，这些物种的净H+排泄是由鳃Na+/H+交换驱动的，NHE3起主导作用，甚至在一些淡水物种中也是如此。本研究的目的有三个方面：1)确定Na+/H+反转运体同源NHE3在窄盐海洋、淡水和全盐硬骨鱼鳃组织中的表达和细胞分布；2)描述系统性酸中毒或外部盐度变化对鳃NHE3表达和分布的影响；3)对鱼类NHE2和NHE3在细胞培养中转染和表达的功能进行分析。为了实现这些目标，他们计划结合分子、生理和免疫学技术来研究鳃转移机制。他们将设计鱼类特异性探针和抗体，并在酸中毒和外部盐度变化后关联体内和细胞变化（mRNA和表达蛋白）。作者还将在转染的哺乳动物成纤维细胞系中功能性表达鱼NHE，并开始体外动力学研究。该项目将为鳃酸碱和离子调控的功能和生理提供新的见解，并可能使我们对脊椎动物NHE亚型的分子结构和保守调控域有更深入的了解。这个为期四年的项目将包括高中生、本科生和理科硕士学生，他们将在夏季期间在家乡机构（佐治亚南方大学）、海洋野外站（Mount Desert Island生物实验室）进行研究，并在约翰霍普金斯大学进行短期的培训访问。

The fish gill carries out the roles of lungs, liver and kidneys in land-dwelling animals. This organ provides the site for gas exchange, metabolic waste excretion, salt balance, and acid-base adjustments. A specific protein in the gill cells is thought to exchange sodium for hydrogen across the membrane of the cells (Na+/H+ exchange; NHE) and in combination with other protein transporters, allows the fish to regulate the levels of acid (pH) in their blood and tissues. NHE has been described in a number of mammalian and non-mammalian systems. These proteins are thought to function in several roles including cell volume regulation, pH balance, and Na+ uptake. Several different types of NHE, each coded by different genes, have been described in mammals. Apically located NHE2 and NHE3 are thought to be specific to transporting epithelial tissues such as kidneys and intestine. In this study, the investigators have focused on the function of the NHE3 antiporter in fish gill and the role this protein plays in acid-base and ion regulation in marine and freshwater fishes.Using molecular biology approaches the investigators have cloned the full length NHE3 transcript in the gills of the marine long-horned sculpin (Myoxocephalus octodecimspinosus). The sculpin NHE3 shows high homology to mammalian NHE3 orthologs. The sequence also includes a variable minisatellite repeat within the coding region. The function (or effect) of this insert is unknown. Northern blots of mRNA for the NHE3 indicated that the 4.5 kb transcript is present in the gills and the level of transcription increases 0.5-2 hours following acidosis. In situ hybridization of gill sections with an NHE3 anti-sense probe identified branchial cells which are thought to also express both NHE2 and Na+/K+-ATPase. Immunological detection of gill NHE3 using a fish specific antibody recognizes a protein on the apical surface of specific gill cells (putative chloride cells) in freshwater adapted Fundulus heteroclitus and the crucian carp (Carassius carassius). The investigators have proposed that net H+ excretion in these species is driven by gill Na+/H+ exchange and that NHE3 plays a predominant role, even in some freshwater species. The aims of this study are three-fold: 1) to determine the presence and cellular distribution of Na+/H+ antiporter ortholog NHE3 expression in gill tissue of a stenohaline marine, freshwater and a euryhaline teleost; 2) to characterize the effects of systemic acidosis, or variations in external salinity on gill NHE3 expression and distribution; 3) to perform a functional analysis of fish NHE2 and NHE3 transfected and expressed in cell culture. To accomplish these goals, they plan to study gill transfer mechanisms using a combination of molecular, physiological, and immunological techniques. They will design fish specific probes and antibodies and correlate in vivo and cellular changes (both mRNA and expressed protein) following acidosis and alterations in the external salinity. The authors will also functionally express the fish NHE in a transfected mammalian fibroblast cell line and begin a study of the kinetics in vitro. This project will provide new insight into the function and physiology of gill acid-base and ion regulation and may lead to a greater understanding of the molecular structure and conserved regulatory domains of vertebrate NHE isoforms. This four year project will involve high school, undergraduate, and Masters of Science students doing research at the home institution (Georgia Southern University), a marine field station (The Mount Desert Island Biological Laboratory) during the summer months, and shorter training visits to Johns Hopkins University.