Investigation of the roles for CaV1.2 in non-excitable tissue during development

研究 CaV1.2 在发育过程中非兴奋组织中的作用

基本信息

批准号：
9348666
负责人：
Geoffrey S Pitt
金额：
$ 45.25万
依托单位：
WEILL MEDICAL COLL OF CORNELL UNIV
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-09-08 至 2021-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9348666
关键词：
Action Potentials Affect Agonist Apoptosis Apoptotic Arrhythmia Autistic Disorder Automobile Driving Behavior Bone Density Bone Development Brain Cells Chondrocytes Clinical Complex Congenital Abnormality Data Development Digit structure Disease Electrophysiology (science)Embryo Endocrine Functional disorder Future Genes Grant Growth Factor Heart Hormones Hyperplasia Hypertrophy Image Internet Investigation Knock-in Knock-out Knowledge Lead Limb structure Location Mandible Membrane Potentials Methods Microscopic Molecular Mosaicism Mus Mutation Neurons Osteoporosis Patients Pattern Pharmacology Phenotype Prevention strategy Proliferating Property Proteins Regulation Rest Role Signal Pathway Signal Transduction Signaling Molecule Skin Speed Syndactyly Technology Testing Timothy syndrome Tissues bone experimental study gain of function gain of function mutation loss of function morphogens mouse model mutant novel osteogenic patch clamp public health relevance response substantia spongiosa targeted agent targeted delivery tool treatment strategy voltage

项目摘要

Although voltage-gated Ca2+ channels (VGCCs) have been studied extensively for their roles in neurons, heart, and hormone-secreting cells, the especially broad array of abnormalities in Timothy syndrome (TS) implies unexpected roles for VGCCs during development. TS is due to a gain-of-function mutation in CACNA1C, the gene encoding the pore-forming α1C subunit of the L-type Ca2+ channel CaV1.2. Intense focus on TS has centered on how the mutant channels lead to cardiac arrhythmias or autism, but almost no studies have examined CaV1.2 contributions to accompanying phenotypes such as syndactyly. Unraveling these CaV1.2 contributions will be fruitful, since these TS phenotypes include multiple common birth defects, implying that dysfunctional CaV1.2 signaling generally is a frequent cause of developmental abnormalities. Moreover, the consequences of dysfunctional CaV1.2 in birth defects demonstrates that CaV1.2 has important, but understudied roles in normal development. Here we propose three Aims: 1: Test the hypothesis that Ca2+ influx through TS mutant CaV1.2 affects interdigital apoptosis; 2: Test the hypothesis that Ca2+ influx through CaV1.2 controls bone development; and 3: Test the hypothesis that the developing mouse limb displays electrical signaling properties that regulate CaV1.2. To complete these aims, we have generated various knockin, knockout, and tissue-specific gain-of-function and loss-of-function mouse models, with which we obtained preliminary data demonstrating roles for CaV1.2 within the developing limb and within developing bone. Further, we have developed methods that build upon multiple technologies to permit live Ca2+ imaging in ex vivo limb cultures isolated from mouse embryos. These methods will provide an unprecedented characterization of parameters such as properties of spontaneous and evoked Ca2+ transients, resting membrane potential, dynamic changes in membrane potential, action potentials, and VGCC currents in developing limbs. Successful completion of these Aims will define novel roles for CaV1.2 in the development of tissues other than brain, heart, and endocrine tissue, thereby revealing mechanisms for many of the unexplained TS phenotypes. Because CaV1.2 anchors a large molecular complex, these studies will serve as a platform for future investigation of the roles of CaV1.2-associated proteins and their downstream signaling partners in development and whether their dysfunction also contributes to birth defects. The availability of clinically used agents that target CaV1.2 offers the opportunity to exploit the knowledge gained from the proposed experiments for new treatment paradigms.

尽管电压门控Ca2+通道（VGCC）已广泛研究其在神经元中的作用，但心脏和分泌细胞，蒂莫西综合征（TS）中特别广泛的异常暗示了VGCC在开发过程中的意外作用。 TS是由于功能收益突变引起的 cacna1c，编码L型Ca2+通道CAV1.2的孔形成α1c亚基的基因。强烈的重点 TS上的集中于突变通道如何导致心律不齐或自闭症，但几乎没有研究已经检查了CAV1.2对参与表型的贡献，例如同步。解开这些 CAV1.2贡献将是富有成果的，因为这些TS表型包括多种常见的先天缺陷，这意味着该功能失调的CAV1.2信号通常通常是发育异常的原因。而且，出生缺陷中功能失调的CAV1.2的后果表明，Cav1.2很重要，但是在正常发展中研究了作用。在这里，我们提出了三个目标：1：检验Ca2+影响的假设通过TS突变体CAV1.2影响二物凋亡； 2：检验Ca2+通过CAV1.2影响的假设控制骨骼发育；和3：检验发育中的鼠标肢体显示电的假设调节CAV1.2的信号传导特性。为了完成这些目标，我们产生了各种敲除，敲除和组织特异性功能获得和功能丧失的小鼠模型，我们获得了初步数据，证明了CAV1.2的作用在发育中的肢体和发育中的骨骼内。此外，我们开发了以多种技术允许从小鼠胚胎分离的离体肢体培养物中实时Ca2+成像。这些方法将为参数提供前所未有的表征，例如赞助和诱发的Ca2+瞬变，静止膜电位，膜电位的动态变化，作用电势和VGCC电流在开发四肢。这些目标的成功完成将定义Cav1.2在组织开发其他方面的新作用而不是大脑，心脏和内分泌组织，从而揭示了许多意外TS的机制表型。由于CAV1.2锚定了大分子络合物，因此这些研究将作为一个平台 CAV1.2相关蛋白质及其下游信号合作伙伴的作用的未来投资发展以及它们的功能障碍是否也导致了先天缺陷。临床使用的可用性针对CAV1.2的代理商提供了探索从提议中获得的知识的机会新治疗范式的实验。