A novel molecular mechanism for stimulating uterine contractility by oxytocin

项目摘要 在怀孕期间，子宫逐渐从静止状态转变，特征是虚弱，不同步， 局部收缩到一种激活状态，在这种状态下，收缩的力度、频率和同步性增加到 在足月时驱逐胎儿。这种转变的一个主要驱动力是子宫肌层平滑肌的逐渐去极化。 细胞(MSMC)膜电位。随着薄膜内部的负电荷变得更少， 子宫肌层变得更容易兴奋。然而，控制这种转变的分子途径尚不清楚， 妨碍我们制定策略来调节子宫收缩能力，以防止早产或产后分娩。 在这里，我们建议检验中心假设，钠(Na)信号复合体由Na-形成。 激活的钾(K)通道SLO2.1和钠泄漏通道NALCN调节这一转变。这 假设是建立在我们从之前的R01获得的已发表和初步数据的基础上。在……里面 足月分离的原代人骨髓间充质细胞，我们发现Na通过NALCN进入激活K外流。 SLO2.1，使膜超极化。接下来，我们展示了这种复合体的激活降低了 子宫条。最后，我们报道了抑制这种复合体诱导MSMC去极化，触发钙 (CA2)通过电压依赖性钙通道进入并促进收缩。总而言之，这些数据表明 NALCN/SLO2.1复合体是控制MSMC膜电位的有力候选者。然而， 因为我们使用的是人体组织，所以我们无法确定这种复合体在逐渐去极化中的作用 妊娠期间MSMC膜电位的变化。为了解决这一限制并充分检验我们的假设，我们的 目的研究NALCN/SLO2.1复合体在小鼠妊娠过程中的功能和调节 MSMC。该项目的目标是：1)确定怀孕期间的NALCN/SLO2.1复杂活动，2)评估 NALCN/SLO2.1复合体活性对细胞内钙离子和子宫收缩功能的影响 MSMC中NALCN/SLO2.1复合体的其他成员，并确定它们对功能的影响 很复杂。为了实现这些目标，我们将定义MSMC膜电位的主要调节器以及如何 随着怀孕的进展，它们会发生变化。这项工作将促进未来旨在开发治疗学的努力 抑制NALCN/SLO2.1复合体以促进分娩或激活复合体以促进静止和 预防早产。

Project Summary During pregnancy, the uterus gradually transitions from a quiescent state characterized by weak, asynchronous, regional contractions to an activated state in which contractions increase in force, frequency, and synchrony to expel the fetus at term. A major driver of this transition is gradual depolarization of the myometrial smooth muscle cell (MSMC) membrane potential. As the inside of the membrane becomes less negatively charged, the myometrium becomes more excitable. However, the molecular pathways controlling this transition are unknown, hampering our ability to develop strategies to regulate uterine contractility to prevent pre- or post-term labor. Here, we propose to test the central hypothesis that a sodium (Na+) signaling complex formed by the Na+- activated potassium (K+) channel SLO2.1 and the Na+ leak channel NALCN regulates this transition. This hypothesis is founded on published and preliminary data we obtained with funding from our previous R01. In primary human MSMCs isolated at term, we showed that Na+ entry through NALCN activated K+ efflux through SLO2.1 and hyperpolarized the membrane. Next, we showed that activation of this complex reduced tension in uterine strips. Finally, we reported that inhibiting this complex induced MSMC depolarization, triggering calcium (Ca2+) entry through voltage-dependent Ca2+ channels and promoting contractility. Together, these data indicate that the NALCN/SLO2.1 complex is a strong candidate to control the MSMC membrane potential. However, because we used human tissues, we could not determine the role of this complex in the gradual depolarization of the MSMC membrane potential over pregnancy. To address this limitation and fully test our hypothesis, our objective is to define the function and regulation of the NALCN/SLO2.1 complex across pregnancy in mouse MSMCs. The goals of this project are to: 1) Define NALCN/SLO2.1 complex activity across pregnancy, 2) Assess the effects of NALCN/SLO2.1 complex activity on intracellular Ca2+ and uterine contractility and 3) Identify additional members of the NALCN/SLO2.1 complex in MSMCs and determine their effects on functionality of the complex. In completing these aims, we will define the main regulators of MSMC membrane potential and how they change as pregnancy progresses. This work will facilitate future efforts aimed at developing therapeutics to inhibit the NALCN/SLO2.1 complex to promote labor or to activate the complex to promote quiescence and prevent preterm labor.