Regulation of calcium concentration in microdomains and primary cilium by TMEM16 proteins and role of TMEM16 and CFTR for renal cystogenesis

细胞内钙浓度的调节在常染色体显性多囊肾病（ADPKD）中起着核心作用，并可能在其他囊性疾病中发挥作用。在ADPKD的发展过程中，囊肿的形成依赖于钙激活的TMEM16氯离子通道。微结构域和初级纤毛是局部钙信号传递的重要结构。我们的初步研究表明，TMEM16通道调节微域内钙浓度。我们的项目将在体内和体外研究TMEM16A和TMEM16F对区室化钙信号的影响。为此，我们将使用肾脏特异性多胱氨酸1 （PC1）敲除（KO）和TMEM16A和TMEM16F双KO模型。TMEM16A和TMEM16B在细胞钙信号传导和囊肿发育中的作用将被研究。我们提出，除了TMEM16通道的功能外，CFTR（囊性纤维化跨膜传导调节因子）的激活对于囊肿的发育是必要的。由于最近在体内发现了TMEM16与CFTR的功能相互作用，因此也将对肾特异性CFTR KO小鼠进行研究。此外，TMEM16对初级纤毛钙信号传导的影响将在体外使用纤毛特异性钙传感器进行分析。由于TMEM16与刺猬信号通路相关，我们也将研究TMEM16在初级纤毛中对该通路的贡献。此外，在收集类导管器官时，将研究初级纤毛、TMEM16和CFTR与囊肿发育的相关性。该项目的结果将阐明TMEM16和CFTR在囊肿发展中的作用。根据预期的发现，可以开发出新的潜在治疗囊肿疾病的方法。

The regulation of the intracellular calcium concentration plays a central role in autosomal dominant polycystic kidney disease (ADPKD) and is potential in other cystic diseases. During the progression of ADPKD the formation of cysts is dependent on calcium activated TMEM16 chloride ion channels. Microdomains as well as the primary cilium are essential structures for local calcium signaling. Our preliminary studies show, that TMEM16 channels modulate the calcium concentration in microdomains. Our project will investigate the impact of TMEM16A and TMEM16F on compartmentalized calcium signaling in vivo and in vitro. For this purpose we will use a kidney specific polycystin1 (PC1) knockout (KO) and TMEM16A and TMEM16F double KO models. The role of TMEM16A and TMEM16B for the cellular calcium signaling and cyst development will be investigated. It is proposed, that in addition to the TMEM16 channels function, activation of CFTR (cystic fibrosis transmembrane conductance regulator) is necessary for cyst development. Because of recently found functional interactions of TMEM16 with CFTR in vivo, kidney specific CFTR KO mice will be investigated, too. Additionally, the impact of TMEM16 on the calcium signaling in the primary cilium will be analyzed in vitro using a cilium specific calcium sensor. As TMEM16 is related to the hedgehog-signaling pathway, the contribution of TMEM16 to this pathway in the primary cilium will be investigated, too. Furthermore, the relevance of the primary cilium, TMEM16 and CFTR for the development of cysts will be investigated in collecting duct organoids. The results of the project will clarify the role of TMEM16 and CFTR for the development of cysts. From the expected findings new potential treatments for cyst diseases could be developed.