Na+/H+ exchange in the nematode intestine

描述（由申请方提供）：Na+/H+交换缺陷损害哺乳动物肠道中的液体和电解质转运。此外，通过H+偶联肽转运蛋白和Na+-葡萄糖共转运蛋白的营养吸收是pH依赖性的，并且可能取决于通过Na+/H +交换的细胞内pH调节以保持活性。虽然大量的是已知的几个Na+/H+交换蛋白的结构和功能，到目前为止，对其余的家庭成员知之甚少。在这个建议中，我们打算开发一个生理学上简单的模型系统，其中单个Na+/H+交换器在介导肠道功能中的作用可以在完整动物的全基因组水平上进行评估。为此，我们克隆了线虫C.优雅本提案的目的1涉及确定这些交换器的细胞、亚细胞和时间表达模式。目的2是直接测试的生理作用，在体内使用双链RNA介导的基因干扰结合一种新的系统来测量细胞内pH值在一个完整的线虫，使用GFP作为靶向的，非侵入性的细胞内pH指示剂。初步结果表明，nhx-2，肠道Na+/H+交换，参与调节早期幼虫期线虫的胚后生长。因此，目标3旨在直接评估这种交换剂在介导营养吸收中的作用。使用转座子插入突变体，缺乏nhx-2基因功能，我们将检查从细菌食物来源的营养物质的摄取，并评估代谢潜力的标志物，如耗氧量，超氧化物的产生，和ATP/ADP浓度。这种多学科的方法将有助于确定个人的Na+/H+交换线虫肠道中的生物学作用。

DESCRIPTION (provided by applicant): Deficiencies in Na+/H+ exchange compromise fluid and electrolyte transport in the mammalian intestine. In addition, nutrient absorption via H+-coupled peptide transporters and Na+-glucose co-transporters is pH dependent, and may depend upon intracellular pH regulation through Na+/H + exchange in order to remain active. Although a great deal is known about the structure and function of several Na+/H+ exchanger proteins, to date little is known about the remaining family members. In this proposal, we intend to develop a physiologically simple model system where the role of individual Na+/H+ exchangers in mediating intestinal function can be assessed on a genome-wide level in an intact animal. Towards this end, we have cloned the cDNAs for nine Na+/H+ exchangers from the nematode C. elegans. Aim 1 of this proposal involves determining the cellular, subcellular and temporal expression patterns of these exchangers. Aim 2 is designed to directly test the physiological role of each exchanger in vivo using double-stranded RNA-mediated gene interference in combination with a novel system to measure intracellular pH in an intact nematode, using GFP as a targetable, non-invasive intracellular pH indicator. Preliminary results suggest that nhx-2, an intestinal Na+/H+ exchanger, is involved in regulating the post-embryonic growth of early larval stage nematodes. Thus, Aim 3 is designed to directly assess the role of this exchanger in mediating nutrient absorption. Using a transposon insertion mutant that lacks nhx-2 gene function, we will examine the uptake of nutrients from a bacterial food source and also assess markers of metabolic potential such as oxygen consumption, superoxide production, and ATP/ADP concentrations. This multidisciplinary approach will help to define the biological role of individual Na+/H+ exchangers in the nematode intestine.