Molecular Physiology of Renal K-Cl Cotransporters

描述（由申请人提供）：偶联的K+-Cl-共转运由KCC蛋白介导，KCC蛋白由SLC 12阳离子-氯化物共转运蛋白基因家族的四个成员编码。基因敲除小鼠的遗传学、生理学和表征已经暗示KCC参与了多种疾病的发病机制，如高血压、癫痫、肾小管酸中毒、神经性疼痛和镰状细胞性贫血。在肾近端小管中，顶端Na吸收诱导的等渗肿胀激活了由KCC 3和KCC 4介导的基底外侧K+ -Cl-共转运，这表明这些转运蛋白参与了过滤后的Na+-Cl-和其他溶质的近端重吸收。事实上，除了减少液体转运，KCC 3缺陷小鼠表现出碳酸氢盐吸收的缺陷，表明KCC 3的损失导致广泛的近端肾小管缺陷。此外，考虑到KCC 3在调节性容量减少（RVD）、对氧化应激的响应和严重的神经退行性综合征中的已证实的作用，我们提出需要KCC 3来维持响应于缺血性容量增加（“IVI”）的细胞完整性，使得KCC 3的损失倾向于缺血性肾小管坏死。因此，近端小管中K+-Cl -共转运的作用和调节是这种竞争性更新的焦点。我们在目标1中提出，除了创建和表征具有KCC 3缺失的小鼠品系（仅限于肾近端小管）之外，还完成表征我们现有的具有KCC 3（Slc 12 a6）基因种系缺失的小鼠品系的肾表型。这些动物研究将包括免疫组织化学、肾生理学和对肾缺血/再灌注损伤反应的评估。 而神经元特异性KCC 2是唯一的介导组成性K+-Cl -共转运，其他三个KCCs是静止的细胞肿胀的情况下。使用嵌合的方法，我们已经本地化的组成性等渗活性的分子决定因素，在C-末端胞质结构域的KCC 2特异性的扩展。考虑到膨胀激活的K +-Cl-协同转运在近端肾小管盐和溶质转运中的作用，我们将使用KCC 4和受抑制的Na-K-2Cl协同转运蛋白NKCC 2之间的嵌合体来表征膨胀激活的分子决定因素。细胞体积对磷酸化状态和膜运输的影响也将在目标2中使用KCC 4和KCC 3的几种N-末端变体进行研究。这些研究将开始在非洲爪蟾卵母细胞，但最终将扩展到负鼠肾（OK）细胞系和/或其他上皮细胞系。在目标3中，我们将集中在保守的半胱氨酸的作用，在机制和调节K+-Cl -共转运，从保守的跨膜半胱氨酸的激活和失活突变的数据的基础上。还将使用各种半胱氨酸耗尽突变体来区分一氧化氮（NO）和相关半胱氨酸反应性化合物对KCC的激活和失活中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.