Mechanisms of Regulation of Anion Exchanger SLC26A6

描述（由申请人提供）： 大部分过滤的CI通过被动和主动途径在近端小管中重吸收。SLC 26 A6最近已成为可能的关键转运蛋白，其解释了该活性组分的重吸收，因此显著促进近端小管NaCl吸收和总体肾NaCl和细胞外容量稳态。本研究的总体目标是阐明调控SLC 26 A6的分子机制。申请人的初步研究表明，在爪蟾卵母细胞中表达的SLC 26 A6的活性被PKC-δ的活化显著抑制，这也改变了其表面表达，而不受PKA活化的影响。当WNK 4在卵母细胞中共表达时，SLC 26 A6也被显著抑制（具有减少的表面和总蛋白表达）。一种密切相关的阴离子转运蛋白Pendrin（SLC 26 A4）不受PKC或WNK 4的影响。 基于这些发现，将追求以下具体目标：1-通过测试PKC-δ特异性肽激活剂和抑制剂的作用，并通过评估PKC-δ是否从胞质溶胶易位到膜，确认PKC-δ是介导SLC 26 A6的PKC调节的PKC同工酶。通过分析SLC 26 A6-Pendrin嵌合转运蛋白，鉴定SLC 26 A6通过PKC对其调控所必需的结构域。2-通过评估PKC活化对MPT细胞（小鼠近端小管细胞系）中内源性表达的SLC 26 A6的活性和表面定位的影响，确认卵母细胞中的发现。确认PKC-δ也是参与的PKC亚型。确认这些细胞中卵母细胞中被鉴定为PKC调节SLC 26 A6活性关键的任何结构域。3-评估WNK 4对SLC 26 A6的抑制性调节是否在翻译或翻译后水平，例如增强的内吞回收和随后的降解靶向。使用与目的1.4类似的方法鉴定WNK 4对其调节所需的SLC 26 A6的结构域-使用介导PKC和WNK 4对其调节的SLC 26 A6的结构域通过酵母双杂交方法鉴定潜在的相关蛋白。通过共沉淀实验确认鉴定的蛋白质与SLC 26 A6的缔合。评估这些蛋白质在介导PKC和WNK 4对SLC 26 A6的调节中的功能作用。

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.