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: 2017
• 资助国家: 加拿大
• 起止时间: 2017-01-01 至 2018-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=630439
• 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.

我的研究项目的中心目标是阐明离子和酸碱调节水生脊椎动物的机制，最终目标是了解从进化到生命周期时间尺度的适应。这包括调查从agnathans到两栖动物的群体。我的研究是综合的，将分子遗传学、生物化学和细胞技术结合到整个动物研究中。这包括强调免疫检测技术的发展和使用，以表征离子转运体的表达，这是我在过去十年中方法论方法的支柱。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和创新探索。这种“泵”具有多个亚基亚型，在不同的分类群中具有不同的进化表达模式。HKA也是胃酸化的核心，并在脊椎动物进化过程中胃丧失的有趣故事中发挥作用。细胞外[K+]的内稳态对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巨大的不良影响。在淡水鱼类中观察到活跃的K+吸收，但其机制尚未明确。我认为HKA是低等脊椎动物钾离子吸收和酸排泄的新机制。有多个HKA a亚基（HKa1, a1b, a2）与HKß或NKß亚基配对作为异源二聚体。我的项目将探讨不同类群中HKa亚单位多样性的生理意义。我将检验多重异构体的表达会导致HKAs的亚功能化和/或新功能化的假设。在只表达一个同工异构体的情况下，我预测一个对偶函数或一个函数的丢失。分类特异性表达模式的复杂模式表明不同的功能模式。鉴于a- β配对在决定转运特性中的重要性，将确定体内和体外表达。胃是脊椎动物的一项重要创新，由酸消化性消化所定义。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胃表型丧失和负责的酸消化基因丧失之间的明确模式。在这一建议中，胃丧失模式下降到家族/属水平将被解决，以确定表型丧失但在离子和/或酸碱调节或新功能募集中保留胃基因的过渡群体。胃酸分泌的成本将使用体内和体外药理学方法进行估计。我将检验高膳食缓冲能力和环境限制Cl是通过降低HKA酸化效果导致胃损失的驱动因素的假设。在鱼类幼虫中，胃的发育可能会延迟数月，因此异时性可能导致幼体发育，呈现出一种有趣的情况，即在酸消化性基因丢失之前，表型可能会丢失。这些基因的命运将被决定。我的NSERC-DG将阐明HKAs在广泛分类群的进化背景下的角色，并阐明其在适应自然或人为环境变化方面的功能重要性。人们将深入了解胃丧失的有趣故事和胃基因的命运。该项目将建立在已有的合作基础上，并创建三个新的合作伙伴，同时为HQP提供强大的培训组成部分，包括为6名研究生和5名本科生提供研究机会。