Characterizing Calcium-Activated Chloride Secretion: Implications for Sex-Linked Variations in Cystic Fibrosis
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Sheridan, John Thomas. Characterizing Calcium-activated Chloride Secretion: Implications for Sex-linked Variations In Cystic Fibrosis. University of North Carolina at Chapel Hill, 2013. https://doi.org/10.17615/pbg3-m139APA
Sheridan, J. (2013). Characterizing Calcium-Activated Chloride Secretion: Implications for Sex-Linked Variations in Cystic Fibrosis. University of North Carolina at Chapel Hill. https://doi.org/10.17615/pbg3-m139Chicago
Sheridan, John Thomas. 2013. Characterizing Calcium-Activated Chloride Secretion: Implications for Sex-Linked Variations In Cystic Fibrosis. University of North Carolina at Chapel Hill. https://doi.org/10.17615/pbg3-m139- Last Modified
- March 22, 2019
- Creator
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Sheridan, John Thomas
- Affiliation: School of Medicine, Department of Cell Biology and Physiology
- Abstract
- Cystic Fibrosis (CF) is a genetic disease that is caused by mutations in the gene encoding the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), the main Cl- channel in human airways, and is characterized by an inability to regulate salt transport in epithelial tissues. In CF lungs, the airway surface liquid (ASL) dehydrates and mucus accumulates, resulting in mucus plugging. This obstructs respiration and leaves CF patients prone to infection. However, the Ca2+-activated Cl- channel (CaCC) is still present and represents an alternative channel to regulate the ASL. Anocatmin 1 (Ano1) was only recently identified as an essential subunit of CaCC and very little is known about Ano1 structure and regulation. In our first study, we utilized several microscopy and biochemistry based techniques to determine the quaternary structure of Ano1 at the plasma membrane. Ano1 was tagged with either mCherry or GFP to conduct Förster resonance energy transfer (FRET) experiments, which revealed that Ano1 oligomerizes prior to reaching the plasma membrane. Additionally, altering the cytoplasmic concentration of Ca2+ did not affect FRET, suggesting a static interaction. Several biochemical assays revealed that Ano1 exists as a dimer. While the incidence of CF is not different between males and females, multiple studies have shown that CF females suffer more severely than their male counterparts. This is most likely due to differences in sex hormones, such as 17β-estradiol (E2). We have previously shown that acute exposure to E2 signals through estrogen receptor alpha (ESR1) to decrease CaCC activity by inhibiting store-operated Ca2+ entry (SOCE). This impinges upon airway secretion and leads to airway dehydration, which may leave female CF patients prone to infection. Therefore, in our second study, we sought to identify the molecular mechanism that is utilized by E2/ESR1 to inhibit SOCE. A key component of SOCE is stromal interaction molecule 1 (STIM1), which oligomerizes and translocates to the endoplasmic reticulum (ER)-plasma membrane junction following ER Ca2+ depletion. Our findings reveal that E2/ESR1 inhibited STIM1 translocation, which correlated with a decrease in STIM1-STIM1 FRET and STIM1 mobility. Additionally, exposure to E2 significantly reduced basal phosphorylation of STIM1, which has been shown to affect function and SOCE. Furthermore, mutating serine 575 to an alanine mimicked the effects of E2. Our data indicate that E2 can signal non-genomically by inhibiting STIM1 phosphorylation, leading to a reduction in SOCE. These findings provide insight into how E2/ESR1 may affect CF and other chronic airway diseases.
- Date of publication
- August 2013
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- In Copyright
- Advisor
- Tarran, Robert
- Degree
- Doctor of Philosophy
- Degree granting institution
- University of North Carolina at Chapel Hill
- Graduation year
- 2013
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