| Summary: | 碩士 === 國立陽明大學 === 解剖學及細胞生物學研究所 === 102 === The ether-à-go-go (Eag) potassium channel belongs to the EAG family of voltage-gated K+ (Kv) channels. Two Eag isoforms have been identified in rat, Eag1 (rEag1) and Eag2. In mammals, the expression of Eag1 potassium channels is neuron-specific and is widely distributed over various brain regions. Despite of their abundant expression in the nervous system, the neurophysiological role of Eag1 potassium channels remains obscure. To better understand the physiological roles of Eag1 channels, we heve applied the yeast two-hybrid screening system to identify rEag1-interacting proteins from a rat brain cDNA library. One of the clones we identified was Mkrn1, an E3 ubiquitin ligase that targets the hTERT (human telomerase reverse transcriptase) for proteasome processing, decreasing telomere activity and subsequently telomere length.
GST pull-down assay and co-immunoprecipitation assay were performed to confirm the interaction between rEag1 and Mkrn1. To investigate whether Mkrn1 functions as an E3 ubiquitin ligase and mediates ubiquitination and degradation of rEag1, cells were treated with cycloheximide (CHX) to inhibit protein synthesis and the degree of rEag1 ubiquitination was exmined. The data demonstrated that Mkrn1 overexpression increased the ubiquitination and decreased the half-life of rEag1 proteins. When co-expressed Mkrn1 and rEag1 in HEK293T cells, in addition to migrating as a doublet, a third low molecular-weight band was detected by Western blotting analysis with anti-rEag1 antibody. Deglycosylation treatment showed that the third band had the molecular weight similar to the deglycosylated form of rEag1, suggesting that Mkrn1 might cause the deglycosylation of rEag1 during the ubiquitination-proteasomal degradation. In addition to the heterologous expression system, we have also performed the immunofluorescence and subcellular fractionation analysis to demonstrate the co-localization of rEag1 and Mkrn1-long in native hippocampal neurons and forebrain lysates.
In this study, we have combined morphological and biochemical methods to investigate the physiological significance of the interaction between Mkrn1-long and rEag1. Together, our data indicates that Mkrn1-long interacts with rEag1 to induce the ubiquitin-mediated degradation of rEag1 K+ channel.
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