Molecular dysregulation of primary cilia TRPP2 channels caused by Finger 1 variants

摘要。常染色体显性多囊肾病（ADPKD）可导致致命的进行性肾衰竭 通常由PKD 2基因的变异引起，PKD 2基因编码TRPP 2离子通道亚基1，2。 尽管我们对ADPKD的遗传基础有很强的理解，但我们仍然不知道TRPP 2是如何影响ADPKD的。 变体影响其离子通道功能5，6。这个基本问题仍然悬而未决，因为TRPP 2定位于 到神秘的初级纤毛--一种微小的天线状细胞器，需要创新的工具来研究。为此， 我们已经开发了新的工具来研究纤毛通道，这些工具可以实时、超原子和原子水平产生结果。 分辨率，从而提供了迄今为止TRPP 2上ADPKD变体的最准确描述。 我们已经发表了使用单粒子cryo-EM 7的TRPP 2的第一个和最高分辨率（3.0 μ m）结构， 这为引起ADPKD的变异体提供了急需的结构背景8. TRPP 2的结构是独特的 在离子通道中，具有称为Top结构域的细胞外盖样组分，其含有59%的 PKD 2基因中发现的种系变异。在Top结构域内是指1基序，其是5 在TRPP 2中发现的最致病的错义变体（R322 Q/W、R325 Q/P和C331 S），因此是TRPP 2中最致病的错义变体。 我们提案的重点8.我们假设，Finger 1变异体将导致TRPP 2功能丧失，原因之一是 两种机制：缺陷TRPP 2通道门控（假设1）或纤毛运输的损失， 通道组装受损（假设2）。在这个建议中，我们设计了三个具体目标来直接测试这些目标。 假设我们将阐明Finger 1变异体对通道生物物理学和纤毛定位的影响 使用目的1中的异源方法。我们将在目标2中使用两种动物模型原位验证这些发现。 此外，我们将测试ADPKD进展的“两次打击”模型，该模型假定该疾病是隐性的 在细胞水平上，只有在剩余的细胞中获得第二次失活体细胞突变后， 等位基因3，4.在目标3中，我们将同时评估变体如何改变TRPP 2通道组装和TRPP 2的表达。 通道门控所需的分子内相互作用。该项目的结果将决定生物物理学 通过Top结构域（该通道的基本和限定组分）调节TRPP 2的调节机制。 通过在分子水平上确定这些变体的影响，我们将打开TRPP 2的大门。 治疗ADPKD 9的靶向合理药物设计。这些之间的潜在机制差异 变异将支持ADPKD个性化药物的基本原理，并通过帮助 来区分致病突变和中性变异。除了ADPKD之外，几种纤毛定位的变体 蛋白质与其他形式的囊性肾病相关10，11。因此，多个肾脏可能 纤毛病变可能共有共同的异常纤毛-细胞信号传导途径，如Ca 2+失调12，13。这些 结果将坚定地确立ADPKD不仅是一种“纤毛病”，而且是一种“通道病”（由 离子通道），其中囊性病变由纤毛的异常Ca 2+信号传导引发14 -17。

ABSTRACT. Autosomal dominant polycystic kidney disease (ADPKD) causes fatal progressive kidney failure and is commonly caused by variants in the PKD2 gene, which encodes the TRPP2 ion channel subunit1,2. Despite our strong understanding of the genetic basis of ADPKD, we still do not know how TRPP2 variants impact its ion channel function5,6. This basic question remains outstanding because TRPP2 localizes to the enigmatic primary cilium—a tiny antenna-like organelle that requires innovative tools to study. To this end, we have developed novel tools to study cilia channels which yield results in real-time, and at super- and atomic resolution, thereby providing the most accurate description to date of ADPKD variants on TRPP2. We have published the first and highest resolution (3.0 Ȧ) structure of TRPP2 using single-particle cryo-EM7, which has provided much-needed structural context for ADPKD-causing variants 8. TRPP2’s structure is unique among ion channels, having an extracellular lid-like component called the Top domain, which contains 59% of the germline variants found in the PKD2 gene. Within the Top domain is the Finger 1 motif, which is the site of 5 of the most pathogenic missense variants found in TRPP2 (R322Q/W, R325Q/P and C331S) and thus is the focus of our proposal8. We hypothesize that Finger 1 variants will cause loss of TRPP2 function due to one of two mechanisms: either defective TRPP2 channel gating (Hypothesis 1) or loss of ciliary trafficking due to impaired channel assembly (Hypothesis 2). In this proposal, we devise three specific aims to directly test these hypotheses. We will elucidate the impact of the Finger 1 variants on channel biophysics and ciliary localization using a heterologous method in Aim 1. We will validate these findings in situ using two animal models in Aim 2. Additionally, we will test the “two-hit” model of ADPKD progression, which posits that this disease is recessive at the cellular level and cysts only develop after acquiring a second inactivating somatic mutation in the remaining allele3,4. In Aim 3, we will concurrently assess how the variants alter TRPP2 channel assembly and the intramolecular interactions required for channel gating. The outcome of this project will determine the biophysical regulatory mechanism(s) of TRPP2 by the Top domain— an essential and defining component of this channel. By determining the impact of these variants at the molecular level, we will open the door to TRPP2- targeted rational drug design for the treatment of ADPKD9. Potential mechanistic differences between these variants would support a rationale for personalized medicine for ADPKD and aid molecular diagnoses by helping to differentiate pathogenic mutations from neutral variants. Beyond ADPKD, variants in several cilia-localized proteins are associated with other forms of cystic kidney diseases10,11. Thus, it is possible that multiple renal ciliopathies may share common aberrant cilia-to-cell signaling pathways, such as Ca2+ dysregulation12,13. These results will firmly establish ADPKD not only as a “ciliopathy” but also as a “channelopathy” (a disease caused by an ion channel), where cystic pathology is initiated by aberrant Ca2+ signaling from the cilium14-17.