Mechanisms of Regulation of Anion Exchanger SLC26A6

阴离子交换剂SLC26A6的调节机制

• 批准号: 7359685
• 负责人: HATIM A HASSAN
• 金额: $ 4.2万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-03-15 至 2008-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7359685
• 关键词: Accounting; Acute; Anions; Apical; Cell Line; Cells; Chimera organism; Complement component C1s; Cyclic AMP-Dependent Protein Kinases; Cytosol; Daily; Employee Strikes; Epithelial; Formates; Goals; Hand; Homeostasis; Hybrids; Isoenzymes; Kidney; Mammalian Cell; Mediating; Membrane; Methods; Molecular; Mus; Nephrons; Oocytes; Oxalates; Pathway interactions; Peptides; Phosphotransferases; Precipitation; Principal Investigator; Protein Isoforms; Protein-Serine-Threonine Kinases; Proteins; Regulation; Retrieval; Role; Signal Pathway; Specificity; Surface; Testing; Urine; Xenopus oocyte; absorption; apical membrane; base; extracellular; inhibitor/antagonist; programs; protein expression; research study; yeast protein

DESCRIPTION (provided by applicant): The bulk of filtered CI is reabsorbed in the proximal tubule by passive and active pathways. SLC26A6 has recently emerged as the likely key transporter that accounts for reabsorption of this active component, and thus contributes significantly to proximal tubule NaCI absorption and overall renal NaCI and extracellular volume homeostasis. The general project goal is to elucidate the molecular mechanisms regulatingSLC26A6. Preliminary studies by the applicant have indicated that activity of SLC26A6 expressed in Xenopus oocytes is significantly suppressed by activation of PKC-delta, which also altered its surface expression, while unaffected by PKA activation. SLC26A6 was also remarkably suppressed (with reduced surface and total protein expression) when WNK4 was co-expressed in oocytes. A closely related anion transporter, Pendrin (SLC26A4), was unaffected by PKC or WNK4. Based on these findings, the following specific aims will be pursued:1- Confirm that PKC-delta is the PKC isozyme mediating PKC regulation of SLC26A6 by testing the effects of PKC-delta specific peptide activator and inhibitor, and by evaluating whether PKC-delta translocates from cytosol to membrane. Identify the domain(s) of SLC26A6 essential for its regulation by PKC by analysis of SLC26A6-Pendrin chimeric transporters. 2- Confirm the findings in oocytes by assessing the effects of PKC activation on activity and surface localization of the endogenously expressed SLC26A6 in MPT cells (a mouse proximal tubule cell line). Confirm that PKC-delta is also the involved PKC isoform. Confirm any domain (s) that is (are) identified as critical for PKC regulation of SLC26A6 activity in oocytes in these cells. 3- Evaluate whether WNK4 suppressive regulation of SLC26A6 is at a translational or a post-translational level, such as enhanced endocytic retrieval and subsequent targeting for degradation. Identify the domains of SLC26A6 required for its regulation by WNK4 using a similar approach as in Aim 1.4- Use the domains of SLC26A6 mediating its regulation by PKC and WNK4 to identify potential associated proteins by the yeast-2-hybrid method. Confirm the association of identified proteins with SLC26A6 by co-precipitation experiments. Evaluate the functional roles of these proteins in mediating regulation of SLC26A6 by PKC and WNK4.

描述（由申请人提供）： 大部分过滤后的 CI 通过被动和主动途径在近端肾小管中被重吸收。 SLC26A6 最近已成为可能的关键转运蛋白，负责该活性成分的重吸收，因此对近端小管 NaCl 吸收以及总体肾脏 NaCl 和细胞外容量稳态有显着贡献。项目的总体目标是阐明调节 SLC26A6 的分子机制。申请人的初步研究表明，非洲爪蟾卵母细胞中表达的SLC26A6的活性被PKC-δ的激活显着抑制，这也改变了其表面表达，同时不受PKA激活的影响。当 WNK4 在卵母细胞中共表达时，SLC26A6 也受到显着抑制（表面和总蛋白表达减少）。密切相关的阴离子转运蛋白 Pendrin (SLC26A4) 不受 PKC 或 WNK4 的影响。 基于这些发现，将追求以下具体目标：1-通过测试 PKC-delta 特异性肽激活剂和抑制剂的作用，并评估 PKC-delta 是否从细胞质转移到细胞膜。通过分析 SLC26A6-Pendrin 嵌合转运蛋白，鉴定 PKC 调节所必需的 SLC26A6 结构域。 2-通过评估 PKC 激活对 MPT 细胞（小鼠近曲小管细胞系）中内源表达的 SLC26A6 的活性和表面定位的影响，确认卵母细胞中的发现。确认 PKC-delta 也是所涉及的 PKC 同工型。确认任何被鉴定为对这些细胞中卵母细胞中 SLC26A6 活性的 PKC 调节至关重要的域。 3-评估SLC26A6的WNK4抑制调节是否处于翻译水平或翻译后水平，例如增强的内吞修复和随后的降解靶向。使用与目标 1.4 中类似的方法识别 WNK4 调节所需的 SLC26A6 结构域 - 使用 SLC26A6 介导 PKC 和 WNK4 调节的结构域，通过酵母 2 混合方法识别潜在的相关蛋白。通过共沉淀实验确认已识别蛋白质与 SLC26A6 的关联。评估这些蛋白质在介导 PKC 和 WNK4 对 SLC26A6 的调节中的功能作用。