Identification and Regulation of the Myometrial Leak Current

描述（由申请人提供）：分娩的开始和成功，足月或早产，取决于有节奏的子宫收缩的增加。宫缩可以通过催产素（OT）治疗来诱导和增强，但近年来，由于其不可预测的结果，OT已被列为高度警惕药物。因此，有必要进一步了解其调节子宫收缩力的基本机制。子宫收缩受子宫肌平滑肌细胞（MSMCs）离子通道活性总和的调控。子宫肌层具有产生节律性收缩的内在能力，其速度依赖于起搏器电位。尽管在20世纪50年代确定了子宫起搏器的潜力，但我们对这种潜力背后的基因和分子机制的理解仍然有限。在心脏和胃肠道中，起搏器电位是由阳离子通过离子通道组合向内泄漏引起的。虽然在肌层中测量到阳离子漏电流，但传导漏电流的通道是未知的。该建议的中心假设是，最近描述的离子通道，钠泄漏通道非选择性（NALCN），在一定程度上是子宫平滑肌细胞中测量的泄漏电流的基础，调节子宫收缩的频率，并由子宫激素包括催产素和乙酰胆碱（ACh）调节。这一假设是基于初步数据，表明NALCN在子宫中表达，并且敲除编码NALCN的基因可以减少MSMCs中的泄漏电流。此外，其他研究表明，包括乙酰胆碱在内的激动剂可以调节NALCN的活性。这一假设将通过以下目标来解决：1)确定NALCN在MSMCs中对漏电流和起搏器电位的贡献程度，并确定其在妊娠期间的调节作用；2)确定子宫素对漏电流活动的影响，并确定相关的信号通路；3)阐明NALCN和漏电流在调节自发性和激动剂诱导的子宫收缩速度中的功能重要性。这些目标将通过结合分子生物学、生物化学、电生理学、药理学、RNAi技术、等距张力记录和体内宫内遥测技术，利用小鼠和人类子宫组织来解决。这项研究具有重要意义，因为其结果将增加我们目前对起搏器潜力的模糊认识，并可能为未来治疗子宫功能障碍提供目标。拟议的研究支持博士前培训计划，该计划还包括课程作业、科学会议和与其他科学家的咨询。这将为申请人在学术界的成功职业生涯做好准备。最后，最终结果将提交到同行评议的期刊上发表。

DESCRIPTION (provided by applicant): The onset and success of labor, at term or preterm, depends on an increase in rhythmic uterine contractions. Contractions can be induced and augmented through oxytocin (OT) treatment, but in recent years, OT has been listed as a High-Alert medication because of its unpredictable outcomes. Therefore, there is a need to better understand its basic mechanisms in regulating uterine contractility. Uterine contractions are regulated by the sum of ion channel activity in myometrial smooth muscle cells (MSMCs). Myometrium possesses an intrinsic ability to produce rhythmic contractions, the pace of which relies on a pacemaker potential. Despite identification of the uterine pacemaker potential in the 1950s, our understanding of the genes and molecular mechanisms that underlie this potential remains limited. In the heart and gastrointestinal tract, pacemaker potentials result from an inward leak of cations through a combination of ion channels. Although a cationic leak current has been measured in the myometrium, the channel that conducts the leak current is unknown. The central hypothesis of this proposal is that a recently described ion channel, the sodium leak channel non-selective (NALCN), in part, underlies the leak current measured in uterine smooth muscle cells, regulates the frequency of uterine contractions, and is modulated by uterotonins including oxytocin and acetylcholine (ACh). This hypothesis is based on preliminary data indicating that NALCN is expressed in the uterus and that knockdown of the gene encoding NALCN reduces the leak current in MSMCs. Additionally, others have shown that NALCN activity can be modulated by agonists, including ACh. This hypothesis will be addressed by pursuing the following aims: 1) determine the extent to which NALCN contributes to the leak current and pacemaker potential in MSMCs and define its regulation throughout pregnancy, 2) determine the effects of uterotonins on leak current activity, and identify the responsible signaling pathway, and 3) elucidate the functional importance of NALCN and the leak current in regulating the pace of spontaneous and agonist-induced uterine contractions. These aims will be addressed by using both mouse and human uterine tissue with a combination of molecular biology, biochemistry, electrophysiology, pharmacology, RNAi technology, isometric tension recordings, and in vivo intrauterine telemetry. The proposed research is significant because the results will add to our currently vague knowledge of the pacemaker potential and may provide targets for future advancements in the treatment of uterine dysfunction. The proposed studies support a predoctoral training plan that also includes coursework, scientific meetings, and consultation with other scientists. This will prepare the applicant for a successful career in academia. Finally, the end results will be submitted for publication in peer-reviewed journals.