Molecular physiology of ion transport in lower vertebrates.

我的研究计划的中心目标是阐明水生脊椎动物中离子和酸碱调节的机制，最终目的是了解从进化到生命周期时间尺度的适应。这涉及到调查从阿格纳森人到两栖动物的群体。我的研究是综合性的，将分子遗传学、生化和细胞技术结合到整个动物研究中。这包括大力强调开发和使用免疫检测技术来表征离子转运体的表达，这一重点在过去十年中一直是我方法论方法的支柱。我的第一个NSERC-DG将是对氢-钾ATPase(HKA)在离子和酸碱调节中的功能作用的原创性和创新性的探索。这种“泵”有多种亚基异构体，在不同的类群中表达的进化模式不同。HKA也是胃酸化的中心，并在将被讨论的脊椎动物进化中胃丢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是必不可少的，微小的变化可能会产生戏剧性的不利影响。淡水鱼对钾的主动吸收已被观察到，但其机制尚未明确。我认为HKA是低等脊椎动物钾吸收和酸排泄的一种新机制。有多个HKAα亚基(HKa1、A1b、a2)与HK？或NK？亚基配对，作为异源二聚体发挥作用。我的节目将探讨不同分类群中Hka亚基多样性的生理意义。我将检验一种假设，即多种异构体的表达导致HKA的亚功能化和/或新功能化。在只表达一种异构体的情况下，我预测有双重功能或失去一种功能。分类群特有的表达模式的复杂模式表明了不同的功能模式。鉴于a-？配对在确定转运特性中的重要性，将确定体内和体外的表达情况。 胃是脊椎动物的一项重要创新，其定义是酸-胃酸消化。令人惊讶的是，尽管它对消化效率做出了贡献，但胃丢失已经独立发生了多次。我最近的进展建立了一个明确的模式，在胃表型丢失和负责酸胃酸基因的丢失之间。在这项建议中，将解决向下至科/属水平的胃损失模式，以便识别表型丧失但胃基因保留在离子和/或酸碱调节或重新招募新功能的过渡组。胃酸分泌的成本将使用体内和体外的药理学方法进行估计。我将通过降低HKA酸化的效果来测试高饮食缓冲能力和环境氯离子限制是导致胃损失的驱动因素的假设。在鱼类幼体中，胃的发育可能延迟数月，因此异时性可能导致童态畸形，呈现出一种有趣的情况，即表型丢失可能先于酸性-胃酸基因丢失。这些基因的命运将被决定。 我的NSERC-DG将阐明香港会计师事务所在多个分类群的进化背景下所扮演的角色，并阐明其在适应其环境中的自然或人为变化方面的重要作用。我们将深入了解有关胃萎缩和胃基因命运的耐人寻味故事。该项目将建立在现有合作的基础上，并创建三个新的合作，同时为HQP提供强大的培训部分，包括为6名研究生和5名本科生提供研究机会。

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.