TRPC6通道激活突变体致病的结构机制研究

Focal and segmental glomerulosclerosis (FSGS) is a common kidney disorder found in clinic, and up to 20% of patients with kidney diseases have been diagnosed to have FSGS. TRPC6 is a receptor-activated non-selective cation channel and broadly expressed in various tissues and involved in many physiological and pathological processes including smooth muscle contraction, renal filtration, tumor development and heart hypertrophy etc. The genetic gain-of-function mutation of human TRPC6 gene is linked to the familial FSGS. It is reported that up to 11 activating mutations of TRPC6 have been identified to cause familial FSGS. Therefore, TRPC6 is an important drug target for the treatment of FSGS-associated kidney diseases. Recently, our group has successfully solved the structure of the human TRPC6 channel in complex with high affinity inhibitor. However, the structural mechanism of how these pathogenic mutations can lead to FSGS remains unclear. Here we will combine the structural study with other research approaches including biochemistry and electrophysiology techniques to explore the structural mechanism of these pathogenic mutations. The study will shed new lights on the structure-based drug discovery on the human TRPC6 ion channels.

局灶节段性肾小球硬化症（FSGS)是一类临床上常见的肾病，超过20%的肾病患者被诊断为FSGS。TRPC6是一种受体激活的非选择性阳离子通道，在多种组织中表达，参与了平滑肌收缩、肾超滤、肿瘤形成、心肌肥大等多种生理及病理过程。此前的研究发现：编码TRPC6蛋白的基因如果发生激活型突变会导致家族性FSGS的发生。目前已发现多达11个TRPC6激活突变体会造成FSGS。因此，TRPC6成为治疗家族性FSGS肾病的潜在药物靶点。申请人所在课题组此前已成功解析了TRPC6通道结合高亲和力抑制剂复合物的结构，但是这些激活型突变引起TRPC6蛋白结构变化并致病的分子机制却仍不明确。因此，申请人将以TRPC6通道激活突变体的结构为研究重点，结合电生理和生物化学等技术手段，探究这些激活突变体致病的结构机制，这将为特异性识别突变体的药物分子设计提供理论基础。

TRPC通道是一类通透钙离子的非选择性阳离子通道，参与了一系列重要的生理过程。其中TRPC6功能获得性突变（GOF）会引发家族局灶节段性肾小球硬化症（FSGS）。我们探究TRPC通道GOF突变体如何导致疾病，以及抑制剂如何发挥作用的。我们解析了人源TRPC3处于高钙和低钙状态下的冷冻电镜结构。给予这些结构以及电生理研究，我们鉴定出TRPC3上的抑制以及激活钙结合位点，这几个位点将细胞内的钙离子浓度与通道的基础活力偶联起来。这些钙结合位点在TRPC6中也是保守存在的。我们发现TRPC6上的GOF突变体会通过别构废除细胞内的抑制效应来激活通道。 我们还解析了人源TRPC6与两个化学式不一样的抑制剂的结构，这两个抑制剂结合在不同的配体结合口袋，揭示了跨膜区不一样的构象，从而为以TRPC6相关疾病（如FSGS）为靶标的结构基础的药物设计提供了基础。