Molecular Physiology of Renal K-CI Cotransporters

描述(申请人提供)：耦合的K+-CI-共转运是由KCC蛋白介导的，由SLC 12阳离子-氯共转运体基因家族的四个成员编码。遗传学、生理学和基因敲除小鼠的特征表明，KCC与高血压、癫痫、肾小管性酸中毒、神经性疼痛和镰状细胞贫血等多种疾病的发病机制有关。在肾近端小管，顶端钠吸收引起的等渗肿胀激活了由KCC3和KCC4介导的基侧K+-Cl-共转运，这意味着这些转运体参与了近端滤过的Na+-Cl-和其他溶质的再吸收。事实上，除了液体转运减少外，KCC3缺陷小鼠对碳酸氢盐的吸收也存在缺陷，这表明KCC3的丢失会导致全身性近端肾小管缺陷。此外，鉴于KCC3在调节性体积减少(RVD)、对氧化应激的反应以及严重的神经退行性综合征中的作用，我们认为KCC3需要维持细胞完整性以应对缺血体积增加(IVI)，从而使KCC3丢失容易导致缺血性肾小管坏死。因此，近端小管K+-Cl-共转运的作用和调节是这一竞争性更新的焦点。在目标1中，我们建议通过KCC3(Slc12a6)基因的胚系缺失来完成我们现有小鼠品系的肾脏表型鉴定，此外，我们还创建并鉴定了一个仅限于肾脏近端小管的KCC3缺失的小鼠品系。这些动物研究将包括免疫组织化学、肾脏生理学和对肾脏缺血/再灌注损伤的反应的评估。 虽然神经元特异性KCC2在介导结构性K+-Cl-共转运方面是独一无二的，但其他三种KCCs在没有细胞肿胀的情况下是静止的。使用嵌合方法，我们已经将构成等张活性的分子决定因素定位为C端细胞质结构域中KCC2特异性的扩展。考虑到溶胀激活的K+-Cl-共转运蛋白在近端肾小管盐分和溶质转运中的作用，我们将利用KCC4和S抑制的Na-K-2Cl共转运蛋白NKCC2之间的嵌合体来表征溶胀激活的分子决定因素。在Aim 2中，也将使用KCC4和KCC3的几个N端变体来研究细胞体积对磷酸化状态和膜转运的影响。这些研究将从非洲爪哇的卵母细胞开始，但最终将扩展到负鼠肾脏(OK)细胞系和/或其他上皮细胞系。在目标3中，我们将基于保守的跨膜半胱氨酸的激活和失活突变的数据，重点研究保守的半胱氨酸在K+-Cl-共转运的机制和调节中的作用。各种半胱氨酸缺失突变体也将被用来区分一氧化氮(NO)和相关的半胱氨酸活性化合物激活和失活KCC的细胞质和膜相关机制。

DESCRIPTION (provided by applicant): Coupled K+-CI- co-transport is mediated by the KCC proteins, encoded by four members of the SLC 12 cation-chloride cotransporter gene family. Genetics, physiology, and the characterization of knockout mice have implicated the KCCs in the pathogenesis of disorders as diverse as hypertension, epilepsy, renal tubular acidosis, neuropathic pain, and sickle cell anemia. In the renal proximal tubule, isotonic swelling induced by apical Na absorption activates basolateral K+ -Cl- cotransport mediated by KCC3 and KCC4, implicating these transporters in proximal re-absorption of filtered Na+-Cl- and other solutes. Indeed, in addition to reduced fluid transport, KCC3-deficient mice exhibit defects in the absorption of bicarbonate, suggesting that loss of KCC3 causes generalized proximal tubular defects. Furthermore, given the demonstrated role of KCC3 in regulatory volume decrease (RVD), the response to oxidant stress, and a severe neurodegenerative syndrome, we propose that KCC3 is required to maintain cellular integrity in response to ischemic volume increase ("IVI"), such that loss of KCC3 predisposes to ischemic tubular necrosis. The role and regulation of K+-Cl - cotransport in the proximal tubule is thus the focus of this competing renewal. We propose in Aim 1 to finish characterizing the renal phenotype of our existing mouse strain with germline deletion of the KCC3 (Slc12a6) gene, in addition to creating and characterizing a mouse strain with KCC3 deletion that is limited to the renal proximal tubule. These animal studies will encompass immunohistochemistry, renal physiology, and assessment of the response to renal ischemia/reperfusion injury. Whereas neuronal-specific KCC2 is unique in mediating constitutive K+-Cl - cotransport, the other three KCCs are quiescent in the absence of cell swelling. Using a chimeric approach, we have localized the molecular determinants of constitutive isotonic activity to a KCC2-specific expansion in the C-terminal cytoplasmic domain. Given the role of swelling-activated K +-Cl- cotransport in proximal tubular salt and solute transport we will characterize the molecular determinants of swelling activation, using chimeras between K CC4 and t he s welling-inhibited N a-K-2Cl cotransporter N KCC2. T he effect o f c ell volume o n phosphorylation status and membrane trafficking will also be studied in Aim 2, using KCC4 and several N-terminal variants of KCC3. These studies will begin in Xenopus oocytes but will ultimately be extended to the opossum kidney (OK) cell line and/or other epithelial cell lines. In Aim 3 we will focus on the role of conserved cysteines in the mechanism and regulation of K+-Cl - cotransport, building on data from both activating and inactivating mutations of conserved transmembrane cysteines. Various cysteine-depleted mutants will also be used to distinguish the cytoplasmic and membrane-associated mechanisms in the activation and inactivation of the KCCs by nitric oxide (NO) and related cysteine-reactive compounds.