Regulation of the epithelial Ca2+ channels TRPV6 and TRPV5

上皮 Ca2 通道 TRPV6 和 TRPV5 的调节

• 批准号: 10538702
• 负责人: Vincenzo Carnevale
• 金额: $ 45.99万
• 依托单位: RUTGERS BIOMEDICAL AND HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-04-01 至 2026-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10538702
• 关键词: Acinus organ component; Acyl Coenzyme A; Binding; Binding Sites; Calmodulin; Charge; Computational Biology; Cryoelectron Microscopy; Development; Docking; Duct (organ) structure; Electrophysiology (science); Enzyme Activation; Enzymes; Epithelial; Equilibrium; Excision; Fluorescence; Funding; Future; Goals; Homology Modeling; Ion Channel; Kidney; Kidney Calculi; Ligands; Lipids; Liquid substance; Lobe; Maps; Measurement; Mediating; Membrane; Molecular; Mutate; Mutation; Mutation Analysis; Pancreas; Pancreatitis; Phosphatidylinositols; Phospholipids; Positioning Attribute; Regulation; Role; Site; Site-Directed Mutagenesis; Specificity; Structure; Surface; Testing; Urine; Work; base; capsaicin receptor; chronic pancreatitis; clinical application; experimental study; fluorescence imaging; insight; loss of function mutation; molecular dynamics; novel therapeutic intervention; premature; prevent; prototype; small molecule; structural biology

ABSTRACT Transient Receptor Vanilloid 6 (TRPV6) and its close relative TRPV5 are Ca2+ selective epithelial ion channels. The membrane phospholipid phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] is the endogenous ligand of these channels that is required for their activity. These channels also undergo Ca2+-induced inactivation which is mediated by binding of Ca2+-calmodulin (CaM) to the channel. TRPV6 and TRPV5 are constitutively active, and their level of activity is determined by the balance between the activating PI(4,5)P2 and the inhibitory CaM. In the previous funding period, we identified the PI(4,5)P2 binding site in TRPV6 using homology modeling and site directed mutagenesis, which was essentially identical to the experimentally determined PI(4,5)P2 binding site in the closely related TRPV5. CryoEM structures for CaM and TRPV6 or TRPV5 also became available, showing a highly consistent picture of one CaM molecule binding to the channel tetramer, and blocking the pore in a fashion consistent with our experimental results. An important development during the previous funding period was the finding that loss of function mutations in TRPV6 are associated with chronic pancreatitis. The likely mechanism is reduced Ca2+ removal from pancreatic fluid in the acini or the ducts leading to increased Ca2+ and premature activation of pancreatic digestive enzymes. Given the crucial role of Ca2+ in digestive enzyme activation, small molecules that increase the activity of TRPV6 can, in principle, be used as novel therapeutic approach to treat pancreatitis. The overall goal of this application is to gain molecular insight into how the two key endogenous regulators PI(4,5)P2 and CaM gate TRPV6 and to use our molecular level understanding of their regulation to identify small molecules that increase their activity. We will use a combination of computational and experimental approaches to decipher the molecular mechanism of PI(4,5)P2 activation of TRPV6 and TRPV5 in Aim1, to determine the relationship between CaM and PI(4,5)P2 regulation of TRPV6 in Aim 2, and to identify small molecules that increase TRPV6 and/or TRPV5 activity by interfering with CaM inhibition in Aim 3. This work will further our understanding of TRPV6 and TRPV5 gating, and small molecules that enhance the activity of TRPV6 or TRPV5 can be used in future experiments to explore the possibility of using this approach to treat or prevent pancreatitis and kidney stones.

摘要 瞬时受体香草素6(TRPV6)及其近亲TRPV5是钙离子选择性上皮离子 频道。膜磷脂磷脂4，5-二磷酸[PI(4，5)P2]是 这些通道的内源性配体，是它们活动所必需的。这些通道还经历了 钙离子引起的失活是通过钙-钙调蛋白(CaM)与通道结合而实现的。 TRPV6和TRPV5在结构上是活性的，它们的活性水平由平衡决定 在激活PI(4，5)P2和抑制CaM之间。在上一个资助期，我们确定了 用同源模建和定点突变的方法对TRPV6中的PI(4，5)P2结合位点进行了分析 与实验测定的PI(4，5)P2结合部位基本相同 TRPV5。用于CaM和TRPV6或TRPV5的低温EM结构也出现了，显示出高度的 一个CaM分子与通道四聚体结合并堵塞通道中的孔洞的一致图片 时尚与我们的实验结果一致。在过去的几年里，一个重要的发展 资助期的研究发现，TRPV6功能突变的丧失与慢性 胰腺炎。其可能机制是腺泡或腺泡内胰液中钙离子的排出减少。 导致钙离子增加和胰腺消化酶过早激活的导管。给定 钙离子在消化酶活性中的关键作用，即增加消化酶活性的小分子 TRPV6原则上可以作为治疗胰腺炎的新方法。总目标 这一应用的目的是从分子上洞察PI(4，5)P2这两个关键的内源性调节因子是如何 和CaM Gate TRPV6，并利用我们对其调控的分子水平的了解来鉴定 增强活性的小分子。我们将结合使用计算和 破译TRPV6和PI(4，5)P2激活分子机制的实验方法 TRPV5在Aim1中的表达，以确定CaM与PI(4，5)P2调节TRPV6在Aim1中的关系 目的2，并鉴定通过干扰TRPV6和/或TRPV5活性增加TRPV6和/或TRPV5的小分子 这项工作将加深我们对TRPV6和TRPV5门控的理解，以及 增强TRPV6或TRPV5活性的小分子可用于未来的实验 探索使用这种方法治疗或预防胰腺炎和肾结石的可能性。