Molecular physiology of ion transport in lower vertebrates.

低等脊椎动物离子运输的分子生理学。

• 批准号: RGPIN-2014-04289
• 负责人: Wilson, Jonathan
• 金额: $ 2.19万
• 依托单位: Wilfrid Laurier University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2014
• 资助国家: 加拿大
• 起止时间: 2014-01-01 至 2015-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=566420
• 关键词: Molecular; physiology; ion; transport; lower

The central objective of my research program is to elucidate the mechanisms of ion and acid-base regulation in aquatic vertebrates with the ultimate goal of understanding adaptation across evolutionary to life-cycle time scales. This involves investigating groups from the agnathans through to the amphibians. My research is integrative, incorporating molecular-genetic, biochemical and cellular techniques to whole animal studies. This includes a strong emphasis on the development and use of immunodetection techniques to characterize the expression of ion transporters, a focus which has served as the backbone of my methodological approach over the past decade. My first NSERC-DG will be an original and innovative exploration into the functional role of the hydrogen-potassium ATPase (HKA) in ion and acid-base regulation. This “pump” has multiple subunit isoforms with diverse evolutionary patterns of expression across taxa. The HKA is also central to gastric acidification, and plays a part in the intriguing story of stomach loss in vertebrate evolution that will be addressed. Homeostasis of extracellular [K+] is essential to control the excitability of nerve and muscle cells and small changes can have dramatic, adverse effects. Active K+ uptake has been observed in freshwater fishes, however, the mechanism has never been defined. I propose the HKA as a novel mechanism for K+ uptake and acid excretion in lower vertebrates. There are multiple HKA a subunits (HKa1, a1b, a2) that pair with HKß or NKß subunits to function as heterodimers. My program will address the physiological significance of the diversity of HKa subunits in different taxonomic groups. I will test the hypothesis that expression of multiple isoforms results in sub-functionalization and/or neofunctionalization of HKAs. In cases where only one isoform is expressed, I predict a dual function or loss of a function. The complex pattern of taxa-specific expression patterns suggests distinct functional patterns. Given the importance of a-ß pairings in determining transport properties, in vivo and ex vivo expression will be determined. The stomach is an important vertebrate innovation that is defined by acid-peptic digestion. Surprisingly, despite its contribution to digestive efficiency, stomach loss has occurred independently multiple times. My recent progress has established a clear pattern between loss of the gastric phenotype and loss of the responsible acid-peptic genes. In this proposal, the stomach loss pattern down to the family/genus level will be resolved in order to identify transition groups with loss of phenotype but with retention of gastric genes in either ion and/or acid-base regulation or recruitment for novel functions. The cost of gastric acid secretion will be estimated using a pharmacological approach in vivo and in vitro. I will test the hypotheses that high dietary buffer capacity, and environmental Cl- limitation are drivers for stomach loss by decreasing the efficacy of HKA acidification. In fish larvae, stomach development may be delayed for months and therefore heterochrony can result in paedomorphosis presenting an intriguing situation of loss of phenotype potentially preceding acid-peptic gene loss. The fate of these genes will be determined. My NSERC-DG will elucidate the role of the HKAs in an evolutionary context across a wide range of taxa and clarify its functional importance to adaptation to natural or anthropogenic changes in their environments. Insights will be gained into the intriguing story of stomach loss and the fate of gastric genes. The project will build upon established collaborations and create three new ones, while providing a strong training component for HQP including research opportunities for 6 graduate and +5 undergraduates.

我的研究计划的中心目标是阐明离子和酸碱调节的水生脊椎动物的最终目标是了解适应跨越进化到生命周期的时间尺度的机制。这涉及到从无颌动物到两栖动物的研究。我的研究是综合性的，将分子遗传学，生物化学和细胞技术纳入整个动物研究。这包括高度重视免疫检测技术的开发和使用，以表征离子转运蛋白的表达，这是我在过去十年中的方法论方法的支柱。我的第一个NSERC-DG将是对氢钾ATP酶（HKA）在离子和酸碱调节中的功能作用的原创性和创新性探索。这种“泵”具有多个亚基同种型，在不同分类群中具有不同的进化表达模式。HKA也是胃酸化的核心，并在脊椎动物进化中胃损失的有趣故事中发挥作用。细胞外[K+]的稳态对于控制神经和肌肉细胞的兴奋性至关重要，微小的变化可能会产生显著的不良影响。淡水鱼对K+的吸收是活跃的，但其吸收机制尚未明确。我建议HKA作为一种新的机制，在较低的脊椎动物的K+吸收和酸排泄。存在多个HKA α亚基（HKa 1、a1 b、a2），其与HKK 1或NK 2亚基配对以作为异源二聚体起作用。我的计划将解决的HKA亚基在不同的分类群体的多样性的生理意义。我将检验多个同种型的表达导致HKA的亚功能化和/或新功能化的假设。在只有一种亚型表达的情况下，我预测双重功能或功能丧失。分类群特异性表达模式的复杂模式表明不同的功能模式。考虑到α-淀粉酶配对在确定转运性质中的重要性，将确定体内和离体表达。胃是一个重要的脊椎动物的创新，是由酸消化的消化。令人惊讶的是，尽管它有助于消化效率，胃损失已经独立发生多次。我最近的进展已经建立了一个明确的模式之间的损失胃表型和损失的负责酸消化基因。在该提案中，将解决科/属水平的胃丢失模式，以识别表型丢失但胃基因保留在离子和/或酸碱调节或招募新功能中的过渡组。将使用体内和体外药理学方法估计胃酸分泌的成本。我将测试的假设，即高饮食缓冲能力，和环境氯限制是胃损失的驱动程序，通过降低HKA酸化的功效。在鱼类幼体中，胃的发育可能会延迟数月，因此异时性可能会导致paedomorphosis呈现出一种有趣的情况，即可能在酸消化基因丢失之前丧失表型。这些基因的命运将被决定。我的NSERC-DG将阐明HKA在广泛的分类群中的进化背景下的作用，并阐明其功能对适应环境中自然或人为变化的重要性。深入了解将获得胃损失和胃基因的命运的有趣的故事。该项目将建立在现有的合作基础上，并创建三个新的合作项目，同时为HQP提供强大的培训组件，包括为6名研究生和5名本科生提供研究机会。