Mechanisms of Regulation of Anion Exchanger SLC26A6

阴离子交换剂SLC26A6的调节机制

• 批准号: 8032670
• 负责人: HATIM A HASSAN
• 金额: $ 5.4万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-03-15 至 2012-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8032670
• 关键词: Accounting; Acute; Anions; Apical; Cell Line; Cells; Chimera organism; Complement component C1s; Cyclic AMP-Dependent Protein Kinases; Cytosol; 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）： 大部分过滤的CI通过被动和活动途径在近端小管中重新吸收。 SLC26A6最近已成为可能重新吸收该活性成分的可能的关键转运蛋白，因此对近端小管NACI吸收以及整体肾脏NACI和细胞外体积稳态有显着贡献。总体项目目标是阐明调节分子机制。申请人的初步研究表明，在异爪蟾卵母细胞中表达的SLC26A6的活性被PKC-DELTA的激活显着抑制，这也改变了其表面表达，而不受PKA激活的影响。当WNK4在卵母细胞中共表达时，SLC26A6也得到了明显抑制（表面和总蛋白表达降低）。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-确认PKC-DELTA是通过测试PKC-DELTA特异性肽激活剂和抑制剂的效果，以及通过评估PKC-DELTA是否从氧化粒酯转换pKC-DELTA，通过测试PKC-DELTA特异性肽激活剂和抑制剂的PKC同工酶介导PKC调节。通过分析SLC26A6-PENDRIN嵌合转运蛋白，确定SLC26A6的域SLC26A6对其对PKC的调节所必需的域。 2-通过评估PKC激活对内源性表达的SLC26A6在MPT细胞中的活性和表面定位的影响来确认卵母细胞中的发现（小鼠近端小管细胞系）。确认PKC-DELTA也是所涉及的PKC同工型。确认（s）被确定为对这些细胞中卵母细胞中SLC26A6活性的PKC调节至关重要的任何域。 3-评估WNK4抑制SLC26A6的抑制性调节是否处于翻译后水平，例如增强的内吞检索和随后的降解靶向。使用与AIM 1.4中的类似方法进行调节所需的SLC26A6域 - 使用SLC26A6的域，通过PKC和WNK4介导其调节的域，通过Yeast-2-Hybrid方法识别潜在的相关蛋白质。通过共沉淀实验确认已鉴定的蛋白与SLC26A6的关联。评估这些蛋白质在PKC和WNK4中介导SLC26A6调节中的功能作用。