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

鱼的鳃在陆生动物身上起着肺、肝和肾的作用。这个器官为气体交换、代谢废物排泄、盐分平衡和酸碱调节提供场所。鳃细胞中的一种特定蛋白质被认为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体结合，使鱼能够调节血液和组织中的酸(PH)水平。它已经在许多哺乳动物和非哺乳动物系统中得到了描述。这些蛋白质被认为具有多种功能，包括调节细胞体积、pH平衡和钠摄取。在哺乳动物中已经描述了几种不同类型的NHE，每种类型都由不同的基因编码。位于顶端的NHE2和NHE3被认为是专用于运输肾脏和肠道等上皮组织的。在这项研究中，研究人员重点研究了NHE3逆向转运蛋白在鱼鳃中的功能以及该蛋白在海洋和淡水鱼酸碱和离子调节中所起的作用。利用分子生物学的方法，研究人员克隆了海洋长角八角鱼(Myoxocephalus Octodecimspinosus)鳃中的全长NHE3转录本。Sculin NHE3与哺乳动物NHE3同源基因高度同源。该序列还包括编码区内的可变小卫星重复序列。此插件的功能(或效果)尚不清楚。NHE3基因的Northern杂交结果表明，4.5kb的转录本存在于鳃中，并且在酸中毒后0.5-2小时内转录水平升高。用NHE3反义探针与鳃切片进行原位杂交，鉴定出同时表达NHE2和Na/K-ATPase的鳃细胞。利用一种鱼特异性抗体对鳃NHE3进行免疫检测，识别淡水适应的异型鳃和鲫鱼(Carassius Carassius)的特定鳃细胞(假定的氯化细胞)顶面上的蛋白质。研究人员提出，这些物种的净H排泄是由鳃的Na/H交换驱动的，NHE3起主导作用，即使在一些淡水物种中也是如此。本研究的目的有三个：1)确定Na/H反向转运蛋白NHE3在狭盐海水、淡水和泛盐硬骨鱼鳃组织中的表达及细胞分布；2)研究全身性酸中毒或外部盐度变化对鱼类NHE3表达和分布的影响；3)对细胞培养中转染和表达的鱼类NHE2和NHE3进行功能分析。为了实现这些目标，他们计划使用分子、生理学和免疫学技术相结合的方法来研究鳃的转移机制。他们将设计鱼类特异的探针和抗体，并将酸中毒和外部盐度变化后体内和细胞的变化(包括mRNA和表达的蛋白质)联系起来。作者还将在转基因的哺乳动物成纤维细胞系中功能性地表达鱼类NHE，并开始体外动力学研究。该项目将为进一步了解脊椎动物NHE异构体的分子结构和保守的调控结构域提供新的视角。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在夏季的几个月里在家庭机构(佐治亚州南方大学)、海洋野外站(沙漠山生物实验室)进行研究，并对约翰·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.