Regulation of an osmosensitive CIC anion channel by Ste20 kinase signaling

描述(申请人提供)：CLC阴离子转运蛋白在从考古细菌到哺乳动物的各种进化生物中表达，它们在不同的过程中扮演着重要的角色，如全身渗透调节以及细胞和细胞器的Cl-和pH的调节。人类九种CLC基因中的五种突变会导致或与遗传性肌肉、骨骼、神经和肾脏疾病有关。尽管进行了大量的研究和生理上的重要性，但对CLCs是如何调节的知之甚少。我们利用线虫秀丽线虫的遗传和分子易操纵性来表征在其天然细胞环境中组装和运行的CLC通道的调节和生理作用。CLH-3b是哺乳动物CLC-1、2、KA和KB阴离子通道亚家族的成员，在线虫卵母细胞中表达，并通过1型磷酸酶介导的丝氨酸去磷酸化被肿胀和减数分裂成熟所激活。Ste20激酶GCK-3与CLH-3b细胞质C末端的101个氨基酸的调节域结合，发挥抑制通道活性的作用。通道抑制需要调节结构域中两个丝氨酸残基的同时磷酸化。我们对CLH-3b的研究提供了该领域中最详细的关于CLC通道调节的描述。GCK-3是哺乳动物Spak和OSR1的同源物。这些激酶已经成为各种运输过程的关键调节者，这些过程在细胞和全身渗透平衡中发挥着重要作用。Spak/OSR1信号通路是抗高血压新药开发的重要靶点。我们已经证明，GCK-3/SPAK/OSR1信号在渗透感受和系统渗透平衡中的作用从线虫到人类都是保守的。这种信号机制在数亿年的进化过程中的功能保守，突显了它的生理意义。这一更新应用建立在DK51610过去的成功和我们对CLH-3b调控的独特理解的基础上，以解决两个基本且尚未解决的问题：信号事件和细胞质C-末端的构象变化如何调制和调节CLC通道属性？细胞和生物体如何检测渗透扰动并将这些变化转化为特定的反应？我们的研究将使用各种分子、电生理和生物物理方法来表征GCK-3和去磷酸化控制CLH-3b活性的信号机制，并表征由磷酸化事件诱导的细胞质C末端和外孔的构象变化。为了充分确定这些蛋白质在生理学和病理生理学中的作用以及它们作为治疗靶点的潜力，对CLC生物学的详细了解是必不可少的。对细胞渗透感觉的分子理解是理解和治疗对人类健康有重大影响的盐和水平衡失调的基石。公共卫生相关性：CLC阴离子转运蛋白执行基本的生理功能，并与人类遗传性肌肉、骨骼、神经和肾脏疾病有关。本申请中描述的研究将首次详细描述磷酸化如何调节CLC通道功能，并将为细胞渗透感觉的机制提供新的见解。对CLC调节和细胞渗透感觉的详细了解对于理解和处理对人类健康有重大影响的盐和水平衡的紊乱是至关重要的。

DESCRIPTION (provided by applicant): ClC anion transport proteins are expressed in evolutionarily diverse organisms ranging from archaebacteria to mammals where they play essential roles in diverse processes such as systemic osmoregulation and regulation of cell and organelle Cl- and pH. Mutations in five of the nine human ClC genes give rise to or are associated with inherited muscle, bone, neurological and kidney disorders. Despite intensive study and their physiological importance, very little is known about how ClCs are regulated. We have exploited the genetic and molecular tractability of the nematode Caenorhabditis elegans to characterize the regulation and physiological roles of ClC channels that are assembled and operational in their native cellular environments. CLH-3b is a member of the mammalian ClC-1, 2, Ka and Kb anion channel subfamily, is expressed in the C. elegans oocyte and is activated by swelling and meiotic maturation via type 1 phosphatase mediated serine dephosphorylation. The Ste20 kinase GCK-3 binds to a 101 amino acid regulatory domain on the CLH-3b cytoplasmic C-terminus and functions to inhibit channel activity. Channel inhibition requires concomitant phosphorylation of two serine residues in the regulatory domain. Our studies of CLH-3b have provided the most detailed description of ClC channel regulation in the field. GCK-3 is a homolog of mammalian SPAK and OSR1. These kinases have emerged as critical regulators of diverse transport processes that play essential roles in cellular and systemic osmotic homeostasis. The SPAK/OSR1 signaling pathway is an important target for the development of new anti- hypertension drugs. We have shown that the role of GCK-3/SPAK/OSR1 signaling in osmosensing and systemic osmotic homeostasis is conserved from C. elegans to humans. The functional conservation of this signaling mechanism over hundreds of millions of years of evolution underscores its physiological significance. This renewal application builds on past successes of DK51610 and our unique understanding of CLH- 3b regulation to address two fundamental and unresolved questions: How do signaling events and conformational changes in the cytoplasmic C-terminus modulate and regulate ClC channel properties? How do cells and organisms detect osmotic perturbations and transduce those changes into specific responses? Our studies will use a variety of molecular, electrophysiological and biophysical approaches to characterize the signaling mechanisms by which GCK-3 and dephosphorylation control CLH-3b activity and to characterize conformational changes in the cytoplasmic C-terminus and outer pore that are induced by phosphorylation events. Detailed understanding of ClC biology is essential in order to fully define the role of these proteins in physiology and pathophysiology and their potential as therapeutic targets. Molecular understanding of cellular osmosensing represents a cornerstone for understanding and treating disturbances of salt and water balance that have a major impact on human health. PUBLIC HEALTH RELEVANCE: ClC anion transport proteins carry out essential physiological functions and are associated with inherited muscle, bone, neurological and kidney disorders in humans. Studies described in this application will provide the first detailed description of how phosphorylation regulates ClC channel function and will provide new insights into mechanisms of cellular osmosensing. Detailed understanding of ClC regulation and cellular osmosensing is essential for understanding and treating disturbances of salt and water balance that have a major impact on human health.