Cilia calcium dysregulation in polycystic kidney disease

摘要。 初级纤毛是一种Ca 2+特权的触角样细胞器，存在于哺乳动物的所有器官系统中。 人体初级纤毛的重要性在越来越多的肾纤毛病中得到了强调- 其中许多是由Ca 2+信号效应基因突变引起的。常染色体显性多囊肾 ADPKD是一种致命的肾纤毛病，可能由PKD 2 Ca 2+通道突变引起。尽管20 自确定其遗传原因以来，我们不知道ADPKD突变如何改变PKD 2通道功能 以及初级纤毛中的Ca 2+失调是否有助于肾囊肿的形成。这些基本问题 因为PKD 2定位于神秘的初级纤毛，这需要创新的工具， study.为此，我们的实验室开发了新的方法来研究睫状体Ca 2+信号和PKD 2通道 ADPKD突变引起的调节异常，以超和原子分辨率进行实时监测。 在集合管纤毛中，PKD 2由内部Ca 2+调节，是双峰的。当睫状体Ca 2+升高时 到微摩尔浓度，PKD 2的开放概率增加（CDM），并随着时间的推移变得脱敏 （CDD）。然而，这两个过程的分子机制是未知的。最近，我们发表了一篇驳斥 PKD 2的C-末端EF手参与通道调节和ADPKD进展的假设。在 我们未发表的初步数据，我们确定了一个新的钙离子结合位点的电压传感器域（VSDCa- 位点），其中几个引起ADPKD的突变聚集。我们推测，该位点的突变可能 导致信道功能的损失或增益，这取决于它们对CDM和CDD的功能影响。我们 已经设计了具体的目标，以确定PKD 2的钙依赖性分子调节，同时评估 ADPKD突变的影响。ADPKD没有药物治愈。因此，评估 突变对于设计未来的ADPKD治疗策略至关重要。我们还将测试“睫状体Ca 2 + 通过评估VSDCa位点突变对纤毛Ca 2+动力学的影响， ADPKD患者细胞中的下游基因表达。然后我们将确定是否纠正PKD 2基因编辑 CRISPR/Cas9可以恢复正常的纤毛Ca 2+和基因转录。提出的目标将考验我们有限的 了解初级纤毛中的通道失调如何引发肾脏囊肿的发展。 除ADPKD外，主要影响其他器官系统的纤毛病变经常表现为肾囊肿 合并症因此，这项建议的结果可能会延伸到其他肾纤毛病变，其中异常 纤毛到细胞的Ca 2+转导是一种可能的统一信号传导机制。

ABSTRACT. The primary cilium is a Ca2+-privileged, antenna-like cellular organelle found in all organ systems of the human body. The importance of primary cilia are highlighted by the growing number of renal ciliopathies— many of which are caused by mutations in Ca2+ signaling effector genes. Autosomal dominant polycystic kidney disease (ADPKD) is a fatal renal ciliopathy that can be caused by mutations in the PKD2 Ca2+ channel. Despite 20 years since determining its genetic cause, we do not know how ADPKD mutations alter PKD2 channel function and if Ca2+ dysregulation in the primary cilium contributes to kidney cyst formation. These basic questions remain outstanding because PKD2 localizes to the enigmatic primary cilium—which requires innovative tools to study. To this end, our lab has developed novel assays to study ciliary Ca2+ signaling and PKD2 channel dysregulation caused by ADPKD mutations in real-time, at super- and atomic resolution. In the cilia of the collecting duct, PKD2 regulated by internal Ca2+ is bimodal. When ciliary Ca2+ is elevated to micromolar concentrations, open probability of PKD2 increases (CDM) and over time becomes desensitized (CDD). However, molecular mechanism for both processes are unknown. Recently, we published a refutation of the hypothesis that C-terminal EF hands of PKD2 is involved in channel regulation and ADPKD progression. In our unpublished preliminary data, we identified a new Ca2+ binding site in the voltage sensor domain (VSDCa- site), where several ADPKD-causing mutations aggregate. We hypothesize that mutations at this site may cause either a loss- or gain-of-channel-function, depending on their functional impact on CDM and CDD. We have devised specific aims to define the Ca2+-dependent molecular regulation of PKD2, while assessing the impact of ADPKD mutations. There is no drug cure for ADPKD. Thus, assessing mechanistic differences between mutations is essential for designing future ADPKD treatment strategies. We will also test the “ciliary Ca2+ hypothesis of cystogenesis” by assessing the impact of VSDCa-site mutations on cilia Ca2+ dynamics and downstream gene expression in ADPKD patient cells. We will then determine if corrective PKD2 gene editing (CRISPR/Cas9) can reinstate normal ciliary Ca2+ and gene transcription. The aims proposed will test our limited understanding of how channel dysregulation in the primary cilia initiates cyst development in the kidney. Beyond ADPKD, ciliopathies which primarily other impact other organ systems, frequently exhibit kidney cysts as comorbidities. Thus, the findings from this proposal may extend to other renal ciliopathies, where aberrant cilia-to-cell Ca2+ transduction is a possible unifying signaling mechanism.