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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：
10011883
负责人：
Geoffrey S Pitt
金额：
$ 47.59万
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/10011883
关键词：
Action Potentials Affect Agonist Apoptosis Apoptotic Arrhythmia 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 autism spectrum disorder 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.

虽然电压门控钙通道（VGCC）在神经元中的作用已被广泛研究，心脏，和分泌细胞，特别是广泛的异常蒂莫西综合征（TS）这意味着VGCC在发育过程中的意外作用。TS是由于一个功能获得性突变， CACNA 1C，编码L型Ca 2+通道CaV1.2的成孔α1C亚基的基因。强烈关注关于TS的研究集中在突变通道如何导致心律失常或自闭症，但几乎没有研究已经研究了CaV1.2对伴随表型如并指畸形的贡献。解开这些 CaV1.2的贡献将是富有成效的，因为这些TS表型包括多种常见的出生缺陷，这意味着 CaV1.2信号传导功能障碍通常是发育异常的常见原因。此外，委员会认为， CaV1.2功能障碍在出生缺陷中的后果表明，CaV1.2具有重要的，但在正常发育过程中的作用。本文提出三个目的：1.验证钙离子内流通过TS突变体CaV1.2影响趾间细胞凋亡; 2：检验Ca 2+通过CaV1.2内流的假设控制骨骼发育;和3：测试发育中的小鼠肢体显示电刺激的假设。调节CaV1.2的信号传导特性。为了完成这些目标，我们已经产生了各种敲入，敲除和组织特异性功能获得和功能丧失小鼠模型，我们获得了初步数据，证明CaV1.2的作用在发育中的肢体和发育中的骨骼中。此外，我们还开发了基于多种技术允许在从小鼠胚胎分离的离体肢体培养物中进行活Ca 2+成像。这些方法将提供参数的前所未有的表征，例如自发和诱发钙瞬变，静息膜电位，膜电位动态变化，动作电位电位和VGCC电流在发展肢体。这些目标的成功完成将定义CaV1.2在其他组织发育中的新作用。比大脑，心脏和内分泌组织，从而揭示了许多原因不明的TS的机制表型由于CaV1.2锚定了一个大的分子复合物，这些研究将作为一个平台， CaV1.2相关蛋白及其下游信号伙伴在他们的功能障碍是否也会导致出生缺陷。临床使用的可用性以CaV1.2为目标的代理提供了利用从建议中获得的知识的机会新的治疗模式的实验。