Molecular mechanisms of osmosensing and signal transduction in C elegans

氯是细胞外液中含量最丰富的阴离子，在许多 必要的生理过程，包括上皮液运输、细胞体积控制、酸碱 动态平衡与细胞兴奋性的调节。阴离子通道介导CI“跨细胞转运和 所有有机体中的细胞器膜。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。 线虫线虫为确定阴离子通道提供了许多实验优势 和CI运输分子生理学。然而，线虫的一个缺点是它的体积小和有限 生理通道。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，我们确定了CIC CI“通道，CLH-3b，该通道 在线虫卵母细胞中表达。CICS在从细菌到动物的生物体中的功能及其 五个人类CIC基因突变的鉴定突显了生理重要性 上升为肾脏、肌肉、骨骼和神经疾病。CLH-3b的生物物理性质类似于 哺乳动物CIC-2的同源性。CLH-3b在卵母细胞减数分裂成熟过程中被去磷酸化激活 肿胀，并将细胞周期进展与排卵相结合。1型磷酸酶CeGLC-7a/p 而一种新发现的Ste20激酶GCK-3调节CLH-3b。GCK-3与CLH-3b结合，失活 通道是一种磷酸化依赖的方式，是哺乳动物PASK的同源基因。PASK规定 Na-K-2CI共转运体参与体液分泌、渗透调节、细胞体积和CI的“内稳态”。 这项续期申请建立在前一笔资金取得相当大的进展和成功的基础上 DK61168循环。在下一个资助期内，我们将结合使用磷酸肽分析， 正向和反向遗传学、电生理学、分子生物学和显微镜1)鉴定CLH-3b 调节磷酸化位点，2)定义CLH-3b调节激酶GCK-3，3)的生理作用 确定调节CLH-3b和整个动物体液平衡的GCK-3信号级联的组件， 和4)开始研究调节clh-3b质膜的机制和基因。 取回。我们建议的研究将为CIC的调控、GCK的功能- 3及其哺乳动物同源物PASK，以及细胞体积感知和 控制细胞CI“含量、上皮液的离子通道和转运体的协调调节 运输和细胞体积。对CIC调节和GCK-3/PASK信号的详细了解是 对于确定阴离子通道在疾病过程中的作用及其潜在的 治疗靶点以及充分认识和治疗液体分泌性疾病。

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.