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

尽管电压门控钙通道(VGCC)在神经元中的作用已被广泛研究， 心脏和激素分泌细胞，这是Timothy综合征(TS)中特别广泛的异常阵列 意味着VGCC在开发过程中扮演了意想不到的角色。TS是由于一种功能增益突变引起的 CACNA1C，编码L型钙通道CaV1.2成孔α1C亚单位的基因。强烈聚焦 On TS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 研究了CaV1.2对伴发表型的贡献，如并指。解开这些 CAV1.2的贡献将是卓有成效的，因为这些TS表型包括多种常见的出生缺陷，这意味着 CaV1.2信号的功能障碍通常是发育异常的常见原因。此外， 功能失调的CaV1.2在出生缺陷中的后果表明，CaV1.2具有重要的 在正常发育中的作用未被充分研究。在这里我们提出了三个目标：1：检验钙离子内流的假设 通过TS突变体CaV1.2影响指间细胞凋亡；2：检验钙通过CaV1.2内流的假设 控制骨骼发育；以及3：测试发育中的小鼠肢体显示电信号的假设 调节CaV1.2的信号特性。 为了实现这些目标，我们产生了各种敲除、敲除和组织特异性功能增益 和功能丧失的小鼠模型，利用这些模型，我们获得了展示CaV1.2作用的初步数据 在发育中的肢体和发育中的骨骼中。此外，我们还开发了基于以下内容的方法 从小鼠胚胎分离的体外肢体培养中允许活体钙成像的多种技术。这些 方法将提供对参数的前所未有的表征，例如自发的和 诱发的钙瞬变，静息膜电位，膜电位的动态变化，作用 在发育中的肢体的电位和VGCC电流。 这些目标的成功完成将定义CaV1.2在其他组织发展中的新角色 而不是大脑、心脏和内分泌组织，从而揭示了许多未解释的TS的机制 表型。由于CaV1.2锚定了一个大的分子复合体，这些研究将成为一个平台 CaV1.2相关蛋白及其下游信号伙伴在细胞周期中的作用 以及他们的功能障碍是否也会导致出生缺陷。临床使用的可获得性 以CaV1.2为目标的代理提供了利用从建议的 新的治疗模式的实验。

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.