Hager, Natalie
(2022)
Development of Fluorogen Activating Protein Tags for Quantitative Trafficking Studies of the Mammalian Potassium Channel Kir2.1 in Saccharomyces cerevisiae.
Doctoral Dissertation, University of Pittsburgh.
(Unpublished)
This is the latest version of this item.
Abstract
Protein composition at the plasma membrane must be tightly regulated for optimal cell health. This involves rapid internalization and selective targeting of proteins to and from the cell surface in response to environmental changes. One category of membrane proteins that are tightly regulated are ion channels, which are dynamically relocalized to or from the plasma membrane in response to physiological changes. This allows organisms to ensure normal ion concentrations. Critical to cardiac function specifically is the localization of Kir2.1, an inward rectifying potassium (Kir) channel, at the cell surface. Kir2.1 restores the resting membrane potential of cardiac cells after each contraction and readies the cells for the next action potential. Defective Kir2.1 trafficking and activity is associated with diseases, including Andersen-Tawil syndrome, highlighting the importance of understanding Kir2.1 trafficking. To elucidate Kir2.1 trafficking, I used a yeast model system where the endogenous potassium channels were deleted and Kir2.1 was expressed and functionality was confirmed using a serial dilution assay. Specifically, I ectopically expressed Kir2.1 in yeast strains that had their endogenous potassium channels, Trk1 and Trk2, deleted and measured growth on low potassium medium. To further evaluate Kir2.1 trafficking I used fluorogen-activating protein (FAP) tags and quantified its cell surface residency. In this document, I include detailed overview of my research on Kir channels and how they are protein trafficked. Next, I describe how we identified the role of specific α-arrestins, an emerging class of protein trafficking adaptors, as regulators of Kir2.1 trafficking. My research demonstrates that a subset of α -arrestins increase intracellular potassium levels by stimulating Kir2.1 trafficking to the cell surface. Following this work, I detail the experiments done to define the trafficking pathways used by the α -arrestin to promote cell surface localization of Kir2.1. Then, I describe the development of the fluorogen-activating protein (FAP) tagging technology and the steps I have taken to make FAP tags more readily available for use in the yeast community. Specifically, I have optimized the tag for expression in yeast, defined optimal imaging conditions, and built a library of FAP-tagged plasmid vectors and proteins to mark specific cellular compartments.
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Details
| Item Type: |
University of Pittsburgh ETD
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| Status: |
Unpublished |
| Creators/Authors: |
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| ETD Committee: |
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| Date: |
11 October 2022 |
| Date Type: |
Publication |
| Defense Date: |
5 July 2022 |
| Approval Date: |
11 October 2022 |
| Submission Date: |
5 August 2022 |
| Access Restriction: |
No restriction; Release the ETD for access worldwide immediately. |
| Number of Pages: |
192 |
| Institution: |
University of Pittsburgh |
| Schools and Programs: |
Dietrich School of Arts and Sciences > Biological Sciences |
| Degree: |
PhD - Doctor of Philosophy |
| Thesis Type: |
Doctoral Dissertation |
| Refereed: |
Yes |
| Uncontrolled Keywords: |
Protein trafficking, yeast genetics, microscopy, Kir2.1, Fluorogen Activating Proteins |
| Date Deposited: |
11 Oct 2022 19:52 |
| Last Modified: |
11 Oct 2022 19:52 |
| URI: |
http://d-scholarship.pitt.edu/id/eprint/43651 |
Available Versions of this Item
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Development of Fluorogen Activating Protein Tags for Quantitative Trafficking Studies of the Mammalian Potassium Channel Kir2.1 in Saccharomyces cerevisiae. (deposited 11 Oct 2022 19:52)
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