Regulation of a C. elegans CIC channel by phosphorylation and membrane retrieval

说明(申请人提供)：氯是细胞外液中含量最丰富的阴离子，在许多基本的生理过程中发挥关键作用，包括上皮液运输、细胞体积控制、酸碱平衡和细胞兴奋性的调节。在所有生物体中，阴离子通道介导CI跨细胞膜和细胞器膜的转运。电生理学研究已经确定了一系列不同的阴离子通道类型，但对阴离子通道的分子生物学和调控知之甚少。线虫为确定阴离子通道和CI转运分子生理学提供了许多实验优势。然而，线虫的一个缺点是它的体积小，生理接触有限。我们最近开发了一些创新的方法，使我们能够绕过这些问题。利用电生理学和反向遗传学的方法，我们鉴定了在线虫卵母细胞中表达的CICCI通道CLH-3b。CICS在从细菌到动物的有机体中的功能，以及它们的生理重要性在五个人类CIC基因突变的确定中得到了强调，这些突变导致了肾脏、肌肉、骨骼和神经疾病。CLH-3b的生物物理性质与哺乳动物的CIC-2相似。CLH-3b在卵母细胞减数分裂成熟和膨胀过程中被去磷酸化激活，其功能是将细胞周期进展连接到排卵。1型磷酸酶CeGLC-7a/p和一种新发现的Ste20激酶GCK-3调节CLH-3b。GCK-3与CLH-3b结合，以磷酸化依赖的方式使该通道失活，是哺乳动物PASK的同源物。PASK调节Na-K-2CI共转运体，参与液体分泌、渗透调节、细胞体积和CI动态平衡。此次续签申请是建立在上一个供资周期DK61168取得重大进展和成功的基础上的。在接下来的资助期间，我们将结合磷酸肽分析、正向和反向遗传学、电生理学、分子生物学和显微镜来1)确定CLH-3b调节磷酸化位点，2)确定CLH-3b调节激酶GCK-3的生理作用，3)确定调节CLH-3b和整个动物体液平衡的GCK-3信号级联反应的组件，以及4)开始表征调节CLH-3b质膜恢复的机制和基因。我们提出的研究将为CIC的调控、GCK-3及其哺乳动物同源蛋白PASK的功能以及基本过程提供重要的新见解，如细胞体积感知以及控制细胞CI含量、上皮液运输和细胞体积的离子通道和转运体的协调调节。对CIC调控和GCK-3/PASK信号的详细了解对于确定阴离子通道在疾病过程中的作用及其作为治疗靶点的潜力以及全面了解和治疗液体分泌性疾病是至关重要的。

DESCRIPTION (provided by applicant): Chloride is the most abundant anion in extracellular fluids and plays critical roles in numerous essential physiological processes including epithelial fluid transport, cell volume control, acid-base homeostasis and regulation of cell excitability. Anion channels mediate CI- transport across cell and organelle membranes in all organisms. Electrophysiological studies have identified a diverse array of anion channel types, but relatively little is known about anion channel molecular biology and regulation. The nematode C. elegans provides numerous experimental advantages for defining anion channel and CI- transport molecular physiology. A drawback of C. elegans, however, is its small size and limited physiological access. We recently developed a number of innovative methods that allow us to circumvent these problems. Using electrophysiology and reverse genetics, we identified a CIC CI- channel, CLH-3b, that is expressed in the C. elegans oocyte. CICs function in organisms from bacteria to animals and their physiological importance is underscored by the identification of mutations in five human CIC genes that give rise to kidney, muscle, bone and neurological disorders. The biophysical properties of CLH-3b resemble those of mammalian CIC-2. CLH-3b is activated by dephosphorylation during oocyte meiotic maturation and swelling, and functions to couple cell cycle progression to ovulation. The type 1 phosphatases CeGLC-7a/p and a newly identified Ste20 kinase, GCK-3, regulate CLH-3b. GCK-3 binds to CLH-3b, inactivates the channel in a phosphorylation dependent manner, and is a homolog of mammalian PASK. PASK regulates Na-K-2CI cotransporters involved in fluid secretion, osmoregulation, and cell volume and CI- homeostasis. This renewal application builds on the considerable progress and successes of the previous funding cycle of DK61168. During the next funding period we will use a combination of phosphopeptide analysis, forward and reverse genetics, electrophysiology, molecular biology and microscopy to 1) identify CLH-3b regulatory phosphorylation sites, 2) define the physiological roles of the CLH-3b regulatory kinase GCK-3, 3) identify components of the GCK-3 signaling cascade that regulates CLH-3b and whole animal fluid balance, and 4) begin characterizing the mechanisms and genes involved in regulating CLH-3b plasma membrane retrieval. Our proposed studies will provide significant new insights into CIC regulation, the function of GCK- 3 and its mammalian homolog PASK, and fundamental processes such as cell volume sensing and the coordinated regulation of ion channels and transporters that control cellular CI- content, epithelial fluid transport and cell volume. Detailed understanding of CIC regulation and GCK-3/PASK signaling is essential in order to define the role of anion channels in disease processes and their potential as therapeutic targets as well as to fully understand and treat fluid secretory diseases.