Identification and Regulation of the Myometrial Leak Current

描述(申请人提供)：分娩的开始和成功，在足月或早产，取决于节律性子宫收缩的增加。催产素(OT)治疗可以诱导和增强宫缩，但近年来，催产素因其不可预测的结果而被列为高警戒药物。因此，有必要更好地了解其调节子宫收缩的基本机制。子宫收缩受子宫肌层平滑肌细胞(MSMC)离子通道活性总和的调节。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的潜力。尽管在20世纪50年代发现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了解仍然有限。在心脏和胃肠道，起搏器电位是由离子通道组合中的阳离子向内泄漏引起的。虽然已经在子宫肌层测量到阳离子泄漏电流，但传导泄漏电流的通道尚不清楚。这一建议的中心假设是，最近被描述的离子通道，即钠泄漏通道非选择性(NALCN)，部分地处于子宫平滑肌细胞中测量的泄漏电流的基础上，调节子宫收缩的频率，并受包括催产素和乙酰胆碱(ACh)在内的子宫紧张素的调节。这一假说基于的初步数据表明，NALCN在子宫中表达，编码NALCN的基因被敲除后，MSMC中的泄漏电流减少。此外，其他研究表明，包括ACh在内的激动剂可以调节NALCN的活性。这一假说将通过以下目标来解决：1)确定NALCN对MSMCs泄漏电流和起搏电位的贡献程度，并确定其在整个妊娠过程中的调节；2)确定子宫内酯对泄漏电流活动的影响，并确定负责的信号通路；以及3)阐明NALCN和泄漏电流在调节自发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、等长张力记录和活体子宫内遥测的小鼠和人类子宫组织来实现。这项拟议的研究意义重大，因为该结果将增加我们目前对起搏器潜力的模糊认识，并可能为未来子宫功能障碍的治疗提供靶点。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。这将为申请者在学术界的成功职业生涯做好准备。最后，最终结果将提交同行评议的期刊发表。

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.