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的离子和酸碱调节机制，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融入到整体动物研究中。这包括高度重视免疫检测技术的开发和使用来表征离子转运蛋白的表达，这一重点在过去十年中一直是我方法论的支柱。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”具有多个亚基亚型，在不同的分类群中具有不同的表达进化模式。 HKA 也是胃酸化的核心，并且在脊椎动物进化过程中胃损失的有趣故事中发挥了作用，这个故事将在下文中讨论。 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巨大的不利影响。在淡水鱼中观察到活性钾的吸收，但其机制尚未明确。我建议 HKA 作为低等脊椎动物吸收钾和排酸的新机制。有多个 HKA a 亚基（HKa1、a1b、a2）与 HKß 或 NKß 亚基配对以充当异二聚体。我的计划将探讨不同分类群中 HKa 亚基多样性的生理意义。我将检验多种亚型的表达导致 HKAs 亚功能化和/或新功能化的假设。在仅表达一种同工型的情况下，我预测会出现双重功能或功能丧失。类群特异性表达模式的复杂模式表明了不同的功能模式。鉴于a-ß配对在确定转运特性中的重要性，将确定体内和离体表达。 胃是脊椎动物的一个重要创新，其定义是胃酸消化。令人惊讶的是，尽管胃损失对消化效率有贡献，但它却多次独立发生。我最近的进展在胃表型丧失和负责胃酸消化基因的丧失之间建立了清晰的模式。在该提案中，将解决下至科/属水平的胃损失模式，以便识别具有表型损失但在离子和/或酸碱调节或招募新功能中保留胃基因的过渡群体。将使用体内和体外药理学方法来估计胃酸分泌的成本。我将检验以下假设：高膳食缓冲能力和环境 Cl- 限制是通过降低 HKA 酸化功效而导致胃损失的驱动因素。在鱼类幼虫中，胃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，即表型丧失可能先于胃酸基因丧失。这些基因的命运将被决定。 我的 NSERC-DG 将阐明 HKA 在各种类群的进化背景中的作用，并阐明其对于适应环境中自然或人为变化的功能重要性。我们将深入了解胃损失和胃基因命运的有趣故事。该项目将以既定的合作为基础，并创建三个新的合作，同时为 HQP 提供强大的培训内容，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.