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    Molecular Determinants of BK Channel Gating and Pharmacology

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    Genre
    Thesis/Dissertation
    Date
    2021
    Author
    Vouga, Alexandre cc
    Advisor
    Rothberg, Brad S.
    Committee member
    Giangiacomo, Kathleen
    Soboloff, Jonathan
    Chong, Parkson Lee-Gau
    Nimigean, Crina
    Department
    Biochemistry
    Subject
    Biochemistry
    Biophysics
    BK channel
    Electrophysiology
    Loperamide
    Permanent link to this record
    http://hdl.handle.net/20.500.12613/6441
    
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    DOI
    http://dx.doi.org/10.34944/dspace/6423
    Abstract
    Large conductance Ca2+-activated K+ channels (BK channels) are expressed ubiquitously in both excitable and non-excitable cells and are important for a range of physiological functions. BK channels gate K+ efflux in response to concurrent depolarized membrane voltage and increased intracellular Ca2+ to modulate action potential shape and duration in neurons, regulate contractility in smooth muscle, and control fluid secretion by epithelial cells in the airway and gut. In addition, mutations in the human BK channel gene (KCNMA1) are linked to neurological disease, such as epilepsy and paroxysmal dyskinesia. Thus, BK channel modulators may provide treatment avenues for BK channelopathies. It will be important to expand our arsenal of BK channel-selective activators and inhibitors and to grow our understanding of their molecular mechanisms of action. Discovery of new channel modulators will further lead to a greater understanding of BK channel structure and function. To better understand the basic structure-function relationship of BK channel gating in response to increased intracellular Ca2+ concentration, in this work I initially investigate structural determinants of BK channel activation in response to conformational changes following Ca2+ binding. I analyze crystal structures of the BK channel cytosolic Ca2+-sensing domain (CSD), also known as the “gating ring”, formed by the C-terminal domains of each of the four identical pore-forming subunits. In the Ca2+-bound state, N449 from the adjacent subunit contacts the bound Ca2+ ion, forming a “Ca2+ bridge.” Mutating N449 to alanine eliminates this coordinate interaction, and using electrophysiology, I found that BK channels with the N449A mutation exhibit a shift in the voltage required for half maximal activation (V1/2) towards more positive voltages. Using size-exclusion chromatography, I observed that the purified BK channel CSD with the N449A mutation shows reduced gating ring oligomerization in response to Ca2+ compared to the wild-type CSD. To further probe molecular determinants of BK channel gating and increase our arsenal of BK channel gating modulators, I optimized a fluorescence-based high throughput screening approach to discover compounds with BK channel inhibitor activity with 99.73% confidence. Through this approach I discovered that the -opioid receptor agonist, loperamide, is a potent BK channel inhibitor. Loperamide (LOP) reduced the open probability of channels at depolarized voltages, but not at very negative voltages when the voltage-sensor is at rest. I observed a weak voltage dependence of loperamide inhibition, consistent with loperamide binding shallow within the inner cavity to block the channel pore. I quantified the inhibitory effect of LOP using an allosteric model in which LOP blocks conduction through open channels and binds with 45-fold higher affinity to the open state over the closed state. These data suggest that loperamide may represent a new class of “use-dependent,” open channel blockers. Together this work describes an approach to understanding BK channel structure and function with the goal of identifying and developing novel therapeutics for the treatment of BK-related diseases.
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