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

抽象的。常染色体显性遗传性多囊肾病(ADPKD)导致致命的进展性肾功能衰竭 通常是由编码TRPP2离子通道亚基1，2的PKD2基因的变异引起的。 尽管我们对ADPKD的遗传基础有了很深的了解，但我们仍然不知道TRPP2是如何 变异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TRPP2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，这些工具产生了实时、超原子的结果 分辨率，从而提供了迄今为止对TRPP2上ADPKD变体的最准确描述。 我们已经发表了第一个也是最高分辨率(3.0Ȧ)的TRPP2结构，使用单粒子低温EM7， 这为导致ADPKD的变体提供了急需的结构背景8.TRPP2的S结构是独一无二的 在离子通道中，有一种细胞外类似盖子的成分，称为Top结构域，它包含59%的 在PKD2基因中发现的生殖系变异。在顶端区域中是Finger 1主题，这是5 在TRPP2(R322Q/W、R325Q/P和C331S)中发现的最具致病性的错义变体之一，因此是 我们建议的重点8。我们假设Finger 1变异会导致TRPP2功能丧失，原因之一是 两种机制：有缺陷的TRPP2通道门控(假设1)或由于以下原因导致的纤毛运输损失 通道组装受损(假设2)。在这项提案中，我们设计了三个具体目标来直接测试这些 假设。我们将阐明Finger 1变体对通道生物物理和纤毛定位的影响 在AIM 1中使用异源方法。我们将在AIM 2中使用两个动物模型在原位验证这些发现。 此外，我们将测试ADPKD进展的“两次打击”模型，该模型假设这种疾病是隐性的 在细胞水平上，只有在获得第二个失活的体细胞突变后才会形成包囊 在目标3中，我们将同时评估这些变体如何改变TRPP2通道组装和 通道门控所需的分子内相互作用。这个项目的结果将决定生物物理 顶级域对TRPP2的调控机制(S)--这是该渠道的一个基本和决定性的组成部分。 通过在分子水平上确定这些变体的影响，我们将打开TRPP2- 治疗ADPKD9的靶向合理药物设计。它们之间的潜在机制差异 变种将支持ADPKD个性化药物的基本原理，并通过帮助 以区分致病突变和中性突变。除ADPKD外，几种纤毛的变异体-本地化 蛋白质与其他形式的囊性肾脏疾病有关10，11。因此，多发性肾脏可能 纤毛病变可能共享共同的异常纤毛到细胞的信号通路，例如钙离子失调12，13。这些 结果将确定ADPKD不仅是一种“睫状病”，而且还是一种“经络病”(一种由 离子通道)，囊性病变是由纤毛14-17发出的异常钙信号引起的。

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.