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 G?q/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.
Public Narrative: Preterm labor, dystocia and post-partum hemorrhage are associated with an increased rate of fetal and maternal morbidities, elevated costs and are due, in part, to, uterine contractile dysfunction. The purpose of this study is to determine how the muscle layer of the uterus becomes more active, and if we can identify specific proteins in the muscle cell layer that regulate this process. From these studies we hope to understand the mechanisms that effectively transition uterine quiescence during pregnancy to contractility at labor.
