Date of Award

2025

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Biomedical Sciences

Track

Molecular and Systems Pharmacology

Research Advisor

Anna Bukiya

Committee

Alex Dopico; Dean Kirson; Helena Parfenova; Valeria Vasquez

Keywords

Cholesterol binding, Equilibrium dialysis, Gating model, nanoDSF, Potassium channel, Steroid

Abstract

Voltage- and calcium-gated, large conductance potassium channels (BK; MaxiK) are ubiquitously expressed and subsequently mediate numerous physiological processes. Cholesterol is consumed in high amounts with the diet and accumulates in cellular membranes when plasma levels become elevated. Cholesterol is a known inhibitor of BK channels and is suspected of contribution to many pathophysiological issues via its interaction with these channels. However, the molecular mechanisms that drive cholesterol inhibition of BK channels remain largely unexplored. This dissertation hypothesized that cholesterol inhibits BK currents via binding to specific amino acid(s) of the channel-forming alpha subunit, and involving distinct modifications of channel gating. This project determines that cholesterol binds to the protein at physiologically relevant levels. A single amino acid, Y450, is required for this interaction between cholesterol and the BK channel-forming alpha subunit, as the conservative mutation of this residue is sufficient to prevent cholesterol binding. While other amino acid residues within the large BK channel-forming protein could interact with cholesterol, determination of their role, if any, requires a separate study. By applying an analytical approach in the form of Horrigan-Aldrich model, this project also identified specific gating parameters that are altered by cholesterol and result in BK channel inhibition. These parameters belong to all three known modes of BK channel gating: intrinsic, calcium-, and voltage-gating. The second major hypothesis of this thesis stated that cholesterol interaction with the BK channel alpha subunit results in cerebral artery diameter physiological responses. Specifically, we addressed the physiological and pharmacological consequences of cholesterol-BK channels protein interaction via Y450. While results demonstrate a lack of change in mouse middle cerebral artery diameter upon cholesterol enrichment, this does not conflict with the hypothesis that cholesterol induces a BK channel-mediated physiological response. Rather, the multimeric nature of BK channel complex and other protein receptors within smooth muscle tissue likely compensates for any alteration of normal cerebral artery physiological action, which is essential to maintaining consistent blood supply to the brain. This study, for the first time, developed a unifying scheme that explains the actions of cholesterol on BK channel alpha subunit function due to direct sterol-protein binding.

ORCID

https://orcid.org/0000-0002-0927-5743

DOI

10.21007/etd.cghs.2025.0689

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