Molecular mechanisms of osmosensing and signal transduction in C elegans

线虫渗透传感和信号转导的分子机制

• 批准号: 8011272
• 负责人: KEVIN STRANGE
• 金额: $ 17.83万
• 依托单位: MOUNT DESERT ISLAND BIOLOGICAL LAB
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-09-01 至 2010-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8011272
• 关键词: Acids; Animals; Anions; Bacteria; Binding; Biological Process; C-terminal; CLH-3B channel; Caenorhabditis elegans; Cell Cycle; Cell Cycle Progression; Cell Volumes; Cell membrane; Cell physiology; Cells; Cellular Stress Response; Chloride Ion; Chlorides; Defect; Development; Disease; Electron Microscopy; Electrophysiology (science); Epithelial; Event; Extracellular Fluid; Fertility; Fluid Balance; Fluids and Secretions; Foundations; Funding; Genes; Genetic; Goals; Homeostasis; Homologous Gene; Human; Immunofluorescence Immunologic; In Vitro; Inherited; Ion Channel; Kidney; Knock-out; Laboratories; Liquid substance; Mediating; Meiosis; Membrane; Methods; Microscopy; Molecular; Molecular Biology; Molecular Genetics; Muscle; Mutagenesis; Mutation; Nematoda; Oocytes; Organelles; Organism; Osmoregulation; Ovulation; Pathway interactions; Phosphopeptides; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphorylation Site; Phosphotransferases; Physiological; Physiological Processes; Physiology; Play; Process; Property; Protein Dephosphorylation; Proteins; Proteolysis; RNA Interference; Regulation; Research; Research Personnel; Retrieval; Role; Serine; Signal Pathway; Signal Transduction; Staging; Surface; Swelling; Testing; Threonine; Transport Process; Ubiquitination; Yeasts; base; bone; genetic analysis; in vitro Assay; innovation; insight; member; nervous system disorder; novel; ovulation time; patch clamp; positional cloning; programs; sodium-potassium chloride cotransporter 2 protein; success; therapeutic target; tool; trafficking; ubiquitin ligase; yeast two hybrid system

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.

氯离子是细胞外液中含量最多的阴离子，在许多细胞中起着至关重要的作用