Signaling Mechanisms in Myometrial Cells that Modulate Na+ Leak Channel Non-Selective (NALCN) during Pregnancy and Labor

摘要 在怀孕结束时，子宫会发生戏剧性的转变，从产生虚弱的、不同步的 宫缩产生强烈的、同步的宫缩，能够分娩胎儿。这一过渡是 通过两种方式产生：1)子宫肌层平滑肌细胞(MSMC)，子宫的肌细胞， 通过缝隙连接相互连接，以及2)MSMC对激动剂的作用变得更加敏感， 如肽物质P，然而，MSMC对其致敏的机制 激动剂还没有完全被理解。这项建议将通过确定机制来解决这一知识差距 通过它来调节离子通道钠泄漏通道非选择性(NALCN)。NALCN感兴趣 因为NALCN传导泄漏电流以使MSMC膜去极化到所需的阈值水平 一种行动潜力。此外，NALCN对于小鼠的电爆发活动和劳动效率也是必需的。 最后，在其他类型的细胞中，收缩诱导激动剂，如P物质，激活NALCN。中环 根据已公布的初步数据进行验证的假设是，在分娩时，辅助蛋白质 Ok80促进NALCN的表达和在质膜上的定位，并使通道增敏 此外，P物质还与G蛋白偶联受体神经激肽受体1和 通过两条信号通路激活NALCN：SRCK和G蛋白GαQ/11。 通过追求两个具体目标来解决。目标1将确定NALCN表达的机制 被上调，该通道定位于质膜并在分娩时激活。两者都是异源的 HEK293细胞和MSMC以及免疫印迹、免疫荧光和电生理的组合 技术将被用来评估UNC80增加表达、膜定位和 NALCN的活性。目标2将确定NALCN对An的作用敏感的机制 分娩中的激动剂。这一目标将通过将HEK293细胞和MSMC与 药理学和电生理学方法。这项拟议的研究意义重大，因为 将揭示一种机制，通过这种机制，MSMC在分娩时对激动剂敏感，为 以开发抑制或增强子宫收缩能力的疗法。拟议的研究支持一项 博士前培训计划，包括课程作业、科学会议、与其他科学家的咨询以及 提交结果，在同行评议的期刊上发表。总体而言，这次经历和培训将为 申请者成功地成为一名从事与女性健康相关研究的学术内科医生和科学家 健康。

ABSTRACT At the end of pregnancy, the uterus makes a dramatic transition from producing weak, non-synchronous contractions to producing strong, synchronous contractions capable of delivering the fetus. This transition is brought about in two ways: 1) the myometrial smooth muscles cells (MSMCs), the muscle cells of the uterus, become interconnected by gap junctions, and 2) the MSMCs become more sensitive to the action of agonists, such as the peptide Substance P. However, the mechanisms by which the MSMCs become sensitized to agonists are not fully understood. This proposal will address this knowledge gap by determining the mechanism by which the ion channel Sodium Leak Channel Non-Selective (NALCN) is regulated. NALCN is of interest because NALCN conducts a leak current to depolarize MSMC membranes to the threshold level needed to elicit an action potential. Additionally, NALCN is required for electrical burst activity and labor efficiency in mice. Finally, in other cell types, contraction-inducing agonists such as Substance P activate NALCN. The central hypothesis, which is founded on published and preliminary data, to be tested is that at labor, the auxiliary protein UNC80 promotes expression of NALCN and localization at the plasma membrane, and sensitizes the channel to Substance P. Furthermore, Substance P binds to the G protein-coupled receptor Neurokinin Receptor 1 and activates NALCN via two signaling pathways: SRC kinase and the G proteins Gαq/11. This hypothesis will be addressed by pursuing two specific aims. Aim 1 will determine the mechanism by which the expression of NALCN is upregulated and the channel is localized to the plasma membrane and activated at labor. Both heterologous HEK293 cells and MSMCs and a combination of immunoblot, immunofluorescence, and electrophysiological techniques will be used to assess the ability of UNC80 to increase expression, membrane localization, and activity of NALCN. Aim 2 will determine the mechanism by which NALCN becomes sensitized to the action of an agonist at labor. This aim will be addressed by using HEK293 cells and MSMCs in combination with pharmacological and electrophysiological approaches. The proposed research is significant because the results will reveal one mechanism by which MSMCs become sensitized to agonists at labor, providing a foundation on which to develop therapeutics to dampen or enhance uterine contractility. The proposed studies support a predoctoral training plan that includes coursework, scientific meetings, consultation with other scientists, and submitting results for publication in peer-reviewed journals. Overall, this experience and training will prepare the applicant for a successful career as an academic physician-scientist conducting research relevant to women's health.