Regulation of the epithelial Ca2+ channels TRPV6 and TRPV5

上皮 Ca2 通道 TRPV6 和 TRPV5 的调节

• 批准号: 10797219
• 负责人: Vincenzo Carnevale
• 金额: $ 8.16万
• 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/10797219
• 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.

摘要 瞬时受体香草酸6（TRPV 6）及其近亲TRPV 5是钙选择性上皮细胞离子 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需的。这些通道也经历 Ca 2+诱导的失活，其通过Ca 2 +-钙调素（CaM）与通道结合介导。 TRPV 6和TRPV 5是组成型活性的，它们的活性水平由TRPV 6和TRPV 5之间的平衡决定。 在活化的PI（4，5）P2和抑制的CaM之间。在上一个融资期，我们确定了 使用同源性建模和定点诱变， 与实验测定的PI（4，5）P2结合位点基本相同， TRPV 5。CaM和TRPV 6或TRPV 5的CryoEM结构也变得可用，显示出高度的特异性。 一个钙调素分子结合到通道四聚体的一致图片，并阻塞了一个通道中的孔。 与我们的实验结果一致。前一个时期的一个重要发展 研究发现TRPV 6的功能缺失突变与慢性胰腺炎相关。 胰腺炎可能的机制是减少了Ca 2+从腺泡或 导管导致Ca 2+增加和胰腺消化酶的过早激活。鉴于 Ca 2+在消化酶激活中的关键作用，增加消化酶活性的小分子， TRPV 6原则上可以作为治疗胰腺炎的新的治疗方法。总目标 本申请的目的是从分子水平深入了解两种关键的内源性调节因子PI（4，5）P2 和CaM门TRPV 6，并利用我们对它们调控的分子水平理解来鉴定 增加其活性的小分子。我们将使用计算和 实验方法来破译PI（4，5）P2激活TRPV 6的分子机制， 在Aim 1中TRPV 5的表达，以确定CaM和PI（4，5）P2调节TRPV 6的关系。 目的2，并鉴定通过干扰TRPV 6和/或TRPV 5活性而增加TRPV 6和/或TRPV 5活性的小分子。 Aim 3中的CaM抑制。这项工作将进一步加深我们对TRPV 6和TRPV 5门控的理解， 增强TRPV 6或TRPV 5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。

项目成果

Particle-based Ising model.

基于粒子的伊辛模型。

• DOI: 10.1103/physreve.103.012125
• 发表时间: 2021
• 期刊: Physical review. E
• 作者: Novinger,Quentin;Suma,Antonio;Sigg,Daniel;Gonnella,Giuseppe;Carnevale,Vincenzo
• 通讯作者: Carnevale,Vincenzo

Protonation underlies tonic vs. use-dependent block.

质子化是强直性阻滞与使用依赖性阻滞的基础。

• DOI: 10.1073/pnas.1802178115
• 发表时间: 2018
• 期刊: Proceedings of the National Academy of Sciences of the United States of America
• 影响因子: 11.1
• 作者: Carnevale,Vincenzo

The DTX Protein Family: An Emerging Set of E3 Ubiquitin Ligases in Cancer.

DTX蛋白家族：癌症中新兴的E3泛素连接酶。

• DOI: 10.3390/cells12131680
• 发表时间: 2023-06-21
• 期刊: CELLS
• 影响因子: 6
• 作者: Scalia, Pierluigi;Williams, Stephen J.;Suma, Antonio;Carnevale, Vincenzo
• 通讯作者: Carnevale, Vincenzo