ORCID

http://orcid.org/0000-0001-8175-9067

Date of Award

Winter 12-15-2021

Author's School

Graduate School of Arts and Sciences

Author's Department

Biology & Biomedical Sciences (Neurosciences)

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

At the end of pregnancy, the uterus transitions from a non-contractile state to a highly contractile state. Two processes primarily drive this transition. First, from the 28th week of pregnancy until labor, the resting membrane potential of uterine (myometrial) smooth muscle cells (MSMCs) gradually becomes more positive (depolarizes) (Parkington et al. 1999). Second, at the end of pregnancy, MSMCs express more oxytocin receptors and become more sensitive to oxytocin (Kimura et al. 1996). However, the detailed mechanisms by which these processes occur have not been determined. My central hypothesis was that the Na+-activated K+ channel SLO2.1 plays a key role in both of these processes. I tested this hypothesis by performing different experiments on primary cultures of MSMCs obtained from samples donated by women undergoing elective C-sections at term non-labor and from an immortalized cell line of human MSMCs (hTERT-HM). First, I confirmed that SLO2.1 channels are expressed in MSMCs and contribute to the resting membrane potential (Ferreira et al., 2019). Second, I showed that activating the oxytocin receptor and protein kinase C (PKC) through the non-canonical pathway inhibits SLO2.1 channels and leads to a depolarization of membrane potential (Ferreira et al., 2019). Third, I showed that the K+ current driven by SLO2.1 channels is modulated by an inward Na+ leak current primarily carried by Na+ leak channel, non-selective (NALCN) (Ferreira et al. 2021 and Ferreira et al. 2019). Fourth, I demonstrated that SLO2.1 and NALCN channels are less than 40 nm from one another in MSMCs and act as a functional complex that can modulate the membrane potential. Activation of this complex promotes membrane hyperpolarization and reduces MSMC excitability and contractility. Finally, I demonstrated that the exact mechanisms are present in mice and showed that the expression and activity of both NALCN and SLO2.1 significantly decrease toward the end of pregnancy. Together, these and additional data support the following model: During the quiescent state, Na+ current through NALCN activates SLO2.1 channels, increasing K+ efflux to maintain MSMCs in a hyperpolarized, non-contractile state. At the end of pregnancy, there is a reduction in the expression of NALCN, leading to decreased SLO2.1 activity. The resulting reduced K+ efflux depolarizes the membrane, leading to activation of voltage-dependent Ca2+ channels (VDCCs), increased intracellular Ca2+, myosin activation, and uterine contractility. Additionally, at labor, oxytocin binds to the oxytocin receptor, leading to the production of inositol triphosphate (IP3) and diacylglycerol (DAG). IP3 activates the release of Ca2+ from intracellular stores, and DAG activates PKC, which inhibits SLO2.1 channels (Ferreira et al., 2019). This SLO2.1 inhibition increases the depolarization of the membrane potential, opens more VDCCs, increases intracellular Ca2+, and activates further the myosin to cause muscle contraction. In addition to significantly increasing our understanding of uterine contractility regulation at pregnancy, my work suggests that SLO2.1 could be an excellent target to regulate uterine activity during pregnancy.

Language

English (en)

Chair and Committee

Celia M. Santi

Committee Members

Jeanne Nerbonne

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