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.