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，子宫从产生微弱的，非同步的 收缩到产生能够分娩胎儿的强有力的同步收缩。这种转型是 通过两种方式产生：1）子宫肌层平滑肌细胞（MSMCs），子宫的肌细胞， 通过间隙连接相互连接，和2）MSMC对激动剂的作用变得更敏感， 然而，MSMCs对肽物质P变得敏感的机制， 激动剂尚未完全了解。该提案将通过确定机制来解决这一知识差距 通过其调节离子通道钠泄漏通道非选择性（NALCN）。NALCN很感兴趣 因为NALCN传导漏电流以使MSMC膜去极化至引发所需的阈值水平， 动作电位此外，NALCN是小鼠的电爆发活动和劳动效率所必需的。 最后，在其他细胞类型中，收缩诱导激动剂如P物质激活NALCN。中央 一个建立在已发表的初步数据基础上的假说有待检验，即在分娩时， UNC80促进NALCN的表达和在质膜上的定位，并使通道敏感 此外，P物质与G蛋白偶联受体神经激肽受体1结合， 通过两条信号通路激活NALCN：SRC激酶和G蛋白G α q/11。这一假设将是 通过追求两个具体目标来解决。目的1将确定NALCN表达的机制， 是上调的，并且该通道定位于质膜并在分娩时被激活。都是异源的 HEK293细胞和MSMCs以及免疫印迹、免疫荧光和电生理学的组合 技术将用于评估UNC80增加表达、膜定位和表达的能力。 NALCN的活动。目的2将确定NALCN对一种药物的作用变得敏感的机制。 分娩时的激动剂。这一目的将通过使用HEK293细胞和MSMCs与 药理学和电生理学方法。这项研究意义重大，因为结果 将揭示MSMCs在分娩时对激动剂敏感的一种机制， 从而开发出抑制或增强子宫收缩力的疗法。拟议的研究支持一项 博士前培训计划，包括课程，科学会议，与其他科学家的咨询， 提交研究结果在同行评审的期刊上发表。总的来说，这种经验和培训将使 申请人成功的职业生涯作为一个学术物理学家，科学家进行有关妇女的研究， 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.