Regulation of the epithelial Ca2+ channels TRPV6 and TRPV5

上皮 Ca2 通道 TRPV6 和 TRPV5 的调节

• 批准号: 10676991
• 负责人: Vincenzo Carnevale
• 金额: $ 48.45万
• 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/10676991
• 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; Epithelium; 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; 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.

抽象的 瞬时受体 Vanilloid 6 (TRPV6) 及其近亲 TRPV5 是 Ca2+ 选择性上皮离子 渠道。膜磷脂磷脂酰肌醇4,5-二磷酸[PI(4,5)P2]是 这些通道的内源性配体是其活性所必需的。这些渠道也经历了 Ca2+ 诱导的失活是通过 Ca2+-钙调蛋白 (CaM) 与通道的结合介导的。 TRPV6和TRPV5具有组成型活性，其活性水平由平衡决定 激活性 PI(4,5)P2 和抑制性 CaM 之间。在上一个资助期间，我们确定了 使用同源建模和定点诱变确定 TRPV6 中的 PI(4,5)P2 结合位点， 与密切相关的实验中确定的 PI(4,5)P2 结合位点基本相同 TRPV5。 CaM 和 TRPV6 或 TRPV5 的冷冻电镜结构也已上市，显示出高度的 一个 CaM 分子与通道四聚体结合并阻塞孔的一致图像 时尚与我们的实验结果一致。上一个时期的一个重要进展 资助期间发现 TRPV6 功能缺失突变与慢性 胰腺炎。可能的机制是腺泡或腺泡中胰液中 Ca2+ 的去除减少。 导管导致 Ca2+ 增加和胰腺消化酶过早激活。鉴于 Ca2+ 在消化酶激活中起着至关重要的作用，小分子可增加消化酶的活性 原则上，TRPV6 可以用作治疗胰腺炎的新方法。总体目标 该应用的目的是从分子角度深入了解两个关键的内源性调节剂 PI(4,5)P2 和 CaM 门 TRPV6 并利用我们对其调节的分子水平理解来识别 增加其活性的小分子。我们将结合使用计算和 破译 PI(4,5)P2 激活 TRPV6 的分子机制的实验方法 Aim1 中的 TRPV5，以确定 TRPV6 中 CaM 和 PI(4,5)P2 调节之间的关系 目标 2，识别通过干扰 TRPV6 和/或 TRPV5 活性的小分子 目标 3 中的 CaM 抑制。这项工作将进一步加深我们对 TRPV6 和 TRPV5 门控的理解，并且 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。