Date of Graduation

2-2026

Document Type

Thesis

Degree Name

Bachelor of Science in Chemistry

Degree Level

Undergraduate

Department

Chemistry & Biochemistry

Advisor/Mentor

Dr. Crystal Archer

Committee Member

Dr. Julie Stenken

Second Committee Member

Dr. Brian Becker

Third Committee Member

Dr. Faith Lessner

Abstract

The epithelial sodium channel (ENaC) plays a critical role in sodium homeostasis, fluid balance, and blood pressure regulation. ENaC activity is tightly controlled by post-translational mechanisms, including ubiquitination-mediated degradation and phosphatidylinositol 4,5-bisphosphate (PIP2) dependent channel activation. PIP2 is a phospholipid necessary to facilitate the maximal channel opening. Previous studies suggest that intracellular PIP2 binding sites are located in proximity to ubiquitination sites on ENaC subunits, raising the possibility of PIP2 binding to influence channel stability and function. This thesis aimed to engineer and validate molecular tools capable of examining the interplay between PIP2 binding and ubiquitination in ENaC regulation. A new alpha-ENaC-Myc plasmid construct was generated and sequence-verified to enable selective biochemical isolation of the channel complex. The existing betaENaC-YFP and gamma-ENaC-pSwick plasmids were used to reconstitute functional ENaC in HEK293T cells, in addition to a plasmid encoding enDUB, a deubiquitinase attached to a nanobody that targets the YFP tag on beta-ENaC. Fluorescence microscopy confirmed robust co-expression of ENaC, establishing a platform compatible with visualization, targeted deubiquitination strategies, and biochemical enrichment. Magnetic bead-based Coimmunoprecipitation (Co-IP) using anti-Myc antibodies was implemented to isolate ENaC and its binding partners under non-denaturing conditions, followed by Western blot analysis to assess ubiquitination and PIP2 association. Preliminary immunoblot results demonstrated successful recovery of alpha-ENaC-Myc, though ubiquitin and PIP2 detection requires further optimization due to the challenges inherent in membrane protein solubilization and lipid-protein interaction preservation. These findings validate the engineered constructs and establish an integrated experimental framework for future quantitative analysis of competitive regulation. Additionally, this work provides a foundational molecular toolkit to investigate how lipid signaling and ubiquitin-dependent pathways coordinately regulate ENaC stability and activity, with implications for understanding ENaC transport and hypertension.

Keywords

ENaC; PIP2; Ubiquitin; PCR; Co-immunoprecipitation; Immunoblot

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