Abstract
Potassium (K(+)) channels are targets of reactive oxygen species in the aging nervous system. KCNB1 (formerly Kv2.1), a voltage-gated K(+) channel abundantly expressed in the cortex and hippocampus, is oxidized in the brains of aging mice and of the triple transgenic 3xTg-AD mouse model of Alzheimer's disease. KCNB1 oxidation acts to enhance apoptosis in mammalian cell lines, whereas a KCNB1 variant resistant to oxidative modification, C73A-KCNB1, is cytoprotective. Here we investigated the molecular mechanisms through which oxidized KCNB1 channels promote apoptosis. Biochemical evidence showed that oxidized KCNB1 channels, which form oligomers held together by disulfide bridges involving Cys-73, accumulated in the plasma membrane as a result of defective endocytosis. In contrast, C73A-mutant channels, which do not oligomerize, were normally internalized. KCNB1 channels localize in lipid rafts, and their internalization was dynamin 2-dependent. Accordingly, cholesterol supplementation reduced apoptosis promoted by oxidation of KCNB1. In contrast, cholesterol depletion exacerbated apoptotic death in a KCNB1-independent fashion. Inhibition of raft-associating c-Src tyrosine kinase and downstream JNK kinase by pharmacological and molecular means suppressed the pro-apoptotic effect of KCNB1 oxidation. Together, these data suggest that the accumulation of KCNB1 oligomers in the membrane disrupts planar lipid raft integrity and causes apoptosis via activating the c-Src/JNK signaling pathway.
Highlights
Oxidation of KCNB1 channels leads to oligomerization and apoptosis
Oxidized KCNB1 Channels Exhibit Defective Endocytosis— Oxidants promote the formation of KCNB1 oligomers that exacerbate apoptosis in mammalian cells [6, 8]
We investigated the molecular mechanisms underlying the cytotoxic effect of KCNB1 oxidation
Summary
Oxidation of KCNB1 channels leads to oligomerization and apoptosis. Results: KCNB1 oligomers aggregate in and disrupt glycolipid raft organization, promoting the activation of the Src/JNK pro-apoptotic pathway. Inhibition of raft-associating c-Src tyrosine kinase and downstream JNK kinase by pharmacological and molecular means suppressed the pro-apoptotic effect of KCNB1 oxidation. Together, these data suggest that the accumulation of KCNB1 oligomers in the membrane disrupts planar lipid raft integrity and causes apoptosis via activating the c-Src/JNK signaling pathway. KCNB1 can be described as a delayed rectifier (non-inactivating) channel, and its oxidation produces two major modifications It induces the oligomerization of the channel through the formation of intersubunit disulfide bridges involving Cys-73 and a cysteine in the C terminus, Cys-710. Cytotoxic Effect of KCNB1 Oxidation raft organization, triggering a stress signal that results in the activation of an apoptotic cascade mediated by c-Src and JNK kinases
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