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Molecular Mechanisms Underlying the Apoptotic Effect of KCNB1 K+ Channel Oxidation

by
Xilong Wu
B.S. Nankai University, China - 2010

Thesis Advisor: Federico Sesti, PhD

Graduate Program in Cellular & Molecular Pharmacology

Thursday, December 5, 2013
1:30 p.m.


Abstract

Oxidative modulation of potassium (K+) channels, critical components for the neuronal excitability in nervous systems, has been implicated in the aging process of nematode Caenorhabditis elegans. During the initiation of my thesis study, we show that KCNB1 potassium channels are direct targets of reactive oxygen species (ROS) in the aging mammalian
brain, and cysteine73 on the channel protein is critical for the formation of channel oligomers via inter-subunit disulfide crosslinks. Moreover, oxidation of wild type KCNB1 enhanced neuronal apoptosis while C73A and C73S mutants confer protective effects. This line of evidence argues that oxidation of KCNB1 may contribute to neurotoxicity in conditions featured by high levels of oxidative stress such as neurodegenerative diseases. Accordingly, native KCNB1 oxidation is significantly exacerbated in the brain of a triple transgenic mouse model of Alzheimer’s disease (AD) (3xTg-AD). Results from the first project suggest that potassium channel oxidation may be a general mechanism in the neurodegeneration of mammalian brain.

In the second project, we found that oxidation-induced KCNB1 oligomers accumulate on the plasma membrane as a result of defective channel internalization (endocytosis) while the C73A mutant shows normal internalization in oxidative conditions. We showed that cholesterol (a primary component of lipid rafts) supplementation reduces KCNB1 oxidation-mediated apoptotic cell death. On the contrary, cholesterol depletion exacerbates the apoptosis in KNCB1-independent manner. Additionally, cholesterol supplementation does NOT rescue defective KCNB1 internalization,
implicating that cholesterol confers viability by restoring the integrity of lipid rafts. Subsequently, we showed that inhibition of c-Src/JNK signaling by molecular and pharmacological approaches prevent apoptotic cell death. Taken together, the second project underscores a model
that accumulation of KCNB1 oligomers on the plasma membrane serves as a transient pro-apoptotic signal which induces apoptotic cell death via disrupting lipid raft integrity and activating c-Src/JNK signaling pathways. Consequently, we also showed that the Cysteine73 is critical for the cellular localization of KCNB1 in the absence of oxidative stress, which may stimulate future investigations on
KCNB1 channel.


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