Acid-activated Cl- channels in endocytic function in kidney and beyond

酸激活的 Cl 通道在肾脏及其他内吞功能中的作用

• 批准号: 527664055
• 负责人: Professor Dr. Thomas J. Jentsch
• 金额: --
• 依托单位: Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP)
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/527664055?language=en
• 关键词: Acid; activated; Cl; channels; endocytic

Luminal ion homeostasis is crucial for vesicular trafficking and function. Important ions are not only H+ (pH), but also luminal Cl-. This is evident from pathologies resulting from disruption of each of the five endolysosomal CLC 2Cl-/H+-exchangers, which we have studied extensively previously. For instance, disruption of ClC-7 leads to osteopetrosis, loss of ClC-3 to severe neurodegeneration, and inactivation of ClC-5 to kidney stones in Dent’s disease. ClC-5 is important for the acidification and Cl- accumulation of apical proximal tubular (PT) endosomes. Without ClC-5, apical endocytosis and recycling of membrane proteins is severely impaired, resulting in proteinuria and secondary changes in Ca2+ metabolism. The same pathology is observed when ClC-5 is converted into an uncoupled Cl conductance in Clcn5 unc mice and in humans carrying similar point mutations. We recently identified Tmem206 as the hitherto enigmatic, broadly expressed acid-activated Cl- channel ASOR. Its role in acidotoxicity is consistent with its partial presence at the plasma-membrane. However, recent work suggests that it is mainly expressed on endosomes, which are normally acidified, and plays a role in endocytic trafficking. We found that ASOR is required for the shrinkage of macropinosomes. More recently we detected abundant ASOR expression in the proximal tubule, where it co-localizes with ClC-5 on apical endosomes. Using our Tmem206 and Clcn5 mouse models, together with acute injection of labelled proteins, or hormones known to induce apical protein recycling, we propose to study endocytic processes in vivo, thus avoiding problems associated with cell culture studies. Exploiting chimeric gene deletion in the same tubule we can compare WT and KO cells side by side under exactly the same conditions. Functional interactions with ClC-5 will be studied in (mostly chimeric) double KO mice comparing all possible cellular genotypes. Further studies will investigate functional interactions with the linear, pH-insensitive endosomal Cl conductance in Clcn5 unc mice. Preliminary results suggest that ASOR KO partially rescues impaired PT endocytosis of Clcn5- mice, suggesting that Tmem206 might be a modifier gene for Dent’s disease. Based on these findings, we will ask whether ASOR disruption alleviates the neurodegeneration associated with Clcn3 disruption. We anticipate that these projects will lead to important insights not only for kidney and CNS physiology and disease, but for endocytosis in general.

腔离子稳态对于囊泡贩运和功能至关重要。重要的离子不仅是H+（pH），还包括腔cl-。这是由于五个内溶性Clc 2cl-/h+-exchangers所产生的病理学来证明的，我们以前已经对此进行了广泛研究。例如，CLC-7的破坏会导致骨质疏松症，CLC-3损失至严重的神经变性，并在Dent病中CLC-5向肾结石失活。 CLC-5对于顶端肾小管（PT）内体的酸化和CL蓄能很重要。没有CLC-5，顶端内吞作用和膜蛋白的回收将严重受损，从而导致蛋白尿和Ca2+代谢的次要变化。当Clc-5转化为CLCN5 UNC小鼠和带有相似点突变的人类中时，观察到相同的病理。我们最近将TMEM206确定为迄今为止的神秘性，广泛表达的酸激活的Cl通道ASOR。它在酸性毒性中的作用与其在血浆膜中的部分存在一致。但是，最近的工作表明，它主要是在通常被酸化的内体上表达的，并且在内吞贩运中起作用。我们发现大肺化体的收缩需要ASOR。最近，我们在近端管中检测到了大量的ASOR表达，并在顶端内体上与CLC-5共定位。我们使用我们的TMEM206和CLCN5小鼠模型，以及急性注射标记的蛋白质或已知可诱导顶蛋白回收的马匹，我们建议在体内研究内吞过程，从而避免与细胞培养研究有关的问题。在同一细胞中利用嵌合基因缺失，我们可以在完全相同的条件下并排比较WT和KO细胞。将研究与CLC-5的功能相互作用，以比较所有可能的细胞基因型（主要是嵌合）双KO小鼠。进一步的研究将研究与CLCN5 UNC小鼠中线性，pH不敏感的内体CL电导的功能相互作用。初步结果表明，ASOR KO部分反应了CLCN5-小鼠的PT内吞作用受损，这表明TMEM206可能是Dent氏病的修饰基因。根据这些发现，我们将询问ASOR中断是否减轻与CLCN3中断相关的神经变性。我们预计，这些项目将不仅可以对肾脏和中枢神经系统的生理学和疾病产生重要的见解，而且对总体内吞作用。