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

鲁米那离子动态平衡对囊泡的运输和功能至关重要。重要的离子不仅是H+(PH)，而且还有流明的Cl-。这一点从我们以前广泛研究的五个内溶酶体Cl2Cl-/H+-交换器的每一个被破坏所引起的病理中可见一斑。例如，在登特氏病中，ClC-7的破坏会导致骨化，ClC-3的丢失会导致严重的神经变性，ClC-5的失活会导致肾结石。ClC-5在顶端近端小管(PT)内小体的酸化和氯离子蓄积过程中起重要作用。如果没有ClC-5，膜蛋白的心尖内吞和循环严重受损，导致蛋白尿和继发性钙代谢改变。在Clcn5 UNC小鼠和携带类似点突变的人类中，当ClC-5被转换为非偶联的Cl电导时，也可以观察到同样的病理。我们最近发现Tem206是迄今为止神秘的、广泛表达的酸激活的氯离子通道ASOR。它在酸中毒中的作用与它在质膜上的部分存在是一致的。然而，最近的研究表明，它主要表达在通常被酸化的内小体上，并在内吞运输中发挥作用。我们发现，ASOR是大管胞缩小所必需的。最近，我们检测到ASOR在近端小管中大量表达，在那里它与顶端内体上的ClC-5共同定位。使用我们的Tmem206和Clcn5小鼠模型，再加上急性注射标记蛋白或已知的诱导顶端蛋白循环的激素，我们建议研究体内的内吞过程，从而避免与细胞培养研究相关的问题。利用同一小管中嵌合基因的缺失，我们可以在完全相同的条件下并排比较WT和KO细胞。与ClC-5的功能相互作用将在(主要是嵌合的)双KO小鼠中进行研究，比较所有可能的细胞基因型。进一步的研究将在Clcn5 UNC小鼠中研究与线性的、pH不敏感的内体氯电导的功能相互作用。初步结果表明，ASOR KO部分挽救了Clcn5-小鼠受损的PT内吞作用，提示Tem206可能是Dent病的修饰基因。基于这些发现，我们将询问ASOR中断是否缓解了与Clcn3中断相关的神经退行性变。我们预计这些项目将不仅对肾脏和中枢神经系统的生理学和疾病，而且对一般的内吞作用带来重要的见解。

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.