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运输和功能至关重要。重要的离子不仅是H+（pH），而且还有鲁米诺Cl-。这是显而易见的病理破坏导致的五个内溶酶体CLC 2Cl-/H+-交换，我们已经广泛研究了以前。例如，ClC-7的破坏导致骨硬化，ClC-3的损失导致严重的神经变性，ClC-5的失活导致登特病中的肾结石。ClC-5对于顶近端小管（PT）内体的酸化和Cl-积累是重要的。没有ClC-5，顶端内吞作用和膜蛋白的再循环严重受损，导致蛋白尿和Ca 2+代谢的继发性变化。当ClC-5在Clcn 5 unc小鼠和携带类似点突变的人类中转化为非偶联Cl电导时，观察到相同的病理学。我们最近将Tmem 206鉴定为迄今为止神秘的、广泛表达的酸激活Cl-通道ASOR。其在酸毒性中的作用与其在质膜上的部分存在一致。然而，最近的工作表明，它主要表达在通常酸化的内体上，并在内吞运输中发挥作用。我们发现ASOR是大胞饮体收缩所必需的。最近，我们在近端小管中检测到丰富的ASOR表达，在那里它与顶端内体上的ClC-5共定位。使用我们的Tmem 206和Clcn 5小鼠模型，连同急性注射标记的蛋白质，或已知诱导顶端蛋白质回收的激素，我们建议在体内研究内吞过程，从而避免与细胞培养研究相关的问题。利用相同小管中的嵌合基因缺失，我们可以在完全相同的条件下并排比较WT和KO细胞。将在（大多为嵌合）双KO小鼠中研究与ClC-5的功能相互作用，比较所有可能的细胞基因型。进一步的研究将调查与Clcn 5 unc小鼠中的线性、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部分挽救受损的PT内吞作用的Clcn 5-小鼠，表明Tmem 206可能是一个修饰基因登特氏病。基于这些发现，我们将询问ASOR中断是否会加剧与Clcn 3中断相关的神经变性。我们预计，这些项目将导致重要的见解，不仅对肾脏和中枢神经系统的生理和疾病，但一般的内吞作用。