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The identification and characterization of disease-associated missense variants in the renal potassium channel ROMK

Nguyen, Nga Hong (2023) The identification and characterization of disease-associated missense variants in the renal potassium channel ROMK. Doctoral Dissertation, University of Pittsburgh. (Unpublished)

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Potassium homeostasis is essential for multiple cellular processes and is vital for maintaining human health. One important regulator of potassium is the renal outer medullary potassium (ROMK) channel, the major potassium secretory channel of the kidney that mediates potassium efflux and provides the driving force for sodium reabsorption. Loss-of-function mutations in ROMK give rise to Bartter syndrome type II, a rare group of disorders characterized by severe electrolyte imbalance and other debilitating symptoms. Moreover, heterozygous carriers with these same mutations have lower blood pressure and are protected from hypertension. Previous work from the Brodsky lab and others have uncovered the underlying molecular mechanisms of several disease-causing ROMK mutations: while some disrupt potassium transport, others impair protein trafficking and led to premature destruction in the endoplasmic reticulum (ER). Yet, a plethora of human polymorphisms in genomic databases, many of which are likely disease-causing, remain undiscovered and uncharacterized. For my dissertation research, I therefore aimed to identify and characterize new disease-linked mutations in ROMK.
Like other membrane proteins, ROMK transits the secretory pathway and must pass quality control checkpoints to achieve its final conformation to function. While several of these pathways have been elucidated, numerous questions remain unanswered. In this document, I first review the pathways governing the trafficking and quality control of potassium channels. Subsequently, I describe my efforts to identify new ROMK disease-associated mutations. Starting with a collaboration with the Bahar lab, I validated the power of a computational method to systematically predict ROMK mutation pathogenicity. Next, with the help of biotech company, Paradigm4, we analyzed human databases and discovered four new disease-associated ROMK mutations. Among these mutations, three led to protein misfolding and premature degradation, while one impaired potassium transport. Finally, I performed random mutagenesis screens to search for hyperactive ROMK mutations, which I predicted would predispose people to hypertension. Data from this study revealed two mutation groups: one that increased steady-state protein levels, and one that slowed current rundown in excised patches. My combined efforts strengthen our understanding of the structure-function relationship of ROMK and outline a pipeline to identify and characterize novel human mutations in a potassium channel.


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Item Type: University of Pittsburgh ETD
Status: Unpublished
CreatorsEmailPitt UsernameORCID
Nguyen, Nga Hongnhn4@pitt.edunhn40000-0002-7629-0601
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairBrodsky, Jeffrey L.jbrodsky@pitt.eduJBRODSKY0000-0002-6984-8486
Committee MemberBerman, Andrea
Committee MemberCarlson, Anne
Committee MemberO'Donnell, Allyson
Committee MemberSubramanya, Arohan
Date: 6 September 2023
Date Type: Publication
Defense Date: 13 July 2023
Approval Date: 6 September 2023
Submission Date: 6 July 2023
Access Restriction: 1 year -- Restrict access to University of Pittsburgh for a period of 1 year.
Number of Pages: 224
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: Mutations that cause disease, potassium transport, kidney disease, human mutations, high blood pressure
Date Deposited: 06 Sep 2023 19:13
Last Modified: 06 Sep 2023 19:13


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