Cilia calcium dysregulation in polycystic kidney disease

抽象的。 初级纤毛是一种钙离子特权的触角状细胞器，存在于所有的器官系统中。 人体。肾脏纤毛疾病的数量不断增加，突显了原发纤毛的重要性。 其中许多是由钙信号效应器基因突变引起的。常染色体显性遗传性多囊肾 疾病(ADPKD)是一种致命的肾脏纤毛疾病，可由PKD2钙通道突变引起。尽管有20个 在确定其遗传原因多年后，我们不知道ADPKD突变是如何改变PKD2通道功能的 如果初级纤毛中的钙离子失调有助于肾囊肿的形成。这些基本问题 仍然很突出，因为PKD2定位于神秘的初级纤毛-这需要创新的工具来 学习。为此，我们实验室开发了新的分析方法来研究睫状肌钙信号和PKD2通道 由ADPKD突变引起的实时、超分辨率和原子分辨率的调节失调。 在集合管纤毛中，受内钙调控的PKD2呈双峰型。睫状肌钙离子升高时 对于微摩尔浓度，PKD2的开放概率增加(CDM)，并随着时间的推移变得不敏感 (CDD)。然而，这两个过程的分子机制尚不清楚。最近，我们发表了一篇驳斥 假设PKD2的C端EF手参与了通道调节和ADPKD的进展。在……里面 在我们未发表的初步数据中，我们在电压传感器结构域(VSDCA-)中发现了一个新的钙结合位点。 站点)，在那里聚集了几个导致ADPKD的突变。我们推测这个位置的突变可能 导致渠道功能丧失或增加，这取决于它们对CDM和CDD的功能影响。我们 已经设计了特定的目标来定义PKD2的钙依赖的分子调控，同时评估 ADPKD突变的影响。ADPKD没有药物治愈的方法。因此，评估两者之间的机制差异 突变对于设计未来的ADPKD治疗策略至关重要。我们还将测试“纤毛钙离子” 通过评估VSDCA位点突变对纤毛钙动态和纤毛生长的影响 ADPKD患者细胞中下游基因的表达。然后我们将确定校正的PKD2基因编辑 (CRISPR/Cas9)可以恢复正常的睫状肌钙离子和基因转录。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的囊性发育。 除ADPKD外，纤毛病变主要影响其他器官系统，经常表现为肾囊肿 作为合并症。因此，这一建议的发现可能延伸到其他异常的肾脏纤毛疾病。 纤毛到细胞的钙离子转导可能是一种统一的信号机制。

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 In our unpublished preliminary data, we identified a new Ca2+ binding site in the voltage sensor domain (VSDCa- site), where several ADPKD-causing mutations aggregate.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.