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研究生:陳宜婷
研究生(外文):CHEN, YI-TING
論文名稱:在神經細胞的大鼠Eag(Ether-a-go-go)鉀離子通道的生化特性
論文名稱(外文):Biochemical Characterization of Rat Eather-a-go-go Potassium Channels in Neurons
指導教授:鄭瓊娟
指導教授(外文):Chung-Jiuan Jeng
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:解剖暨細胞生物學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:63
中文關鍵詞:Eag鉀離子通道海馬迴神經細胞免疫螢光染色溶解度免疫沉澱
外文關鍵詞:Eag potassium channelHippocampal neuronImmunofluorescenceSolubilityImmunoprecipitation
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電位控制鉀離子通道(voltage-gated K+ channel)的基本架構,是由四個鉀離子通道次單位(subunit)所組成的同質或異質(homotetramer or heterotetramer)四聚體。Eag鉀離子通道屬於電位控制鉀離子通道大家族之一,廣泛表現在整個中樞神經系統中。在大鼠Eag鉀離子通道次家族中包含兩個成員:rEag1與rEag2。根據之前研究指出,rEag1與rEag2在培養的海馬迴神經細胞中之次細胞分佈(subcellular localization)情形不同:rEag2主要分布在細胞本體與近端樹突;rEag1除了出現在細胞本體和近端樹突之外,還廣泛的分布於遠端樹突。此外,rEag1最特別的是在樹突上還具有點狀分布的特性。本篇論文的目的是要了解rEag1和rEag2鉀離子通道蛋白的生物化學特性,我們特別是想探究rEag1和rEag2在大鼠前腦(forebrain)及海馬迴(hippocampus)組織中形成herterotetramer和homotetramer的可能性,以及此現象是否可增加rEag鉀離子通道在次細胞分布情形的多樣性。
我們首先觀察在不同培養天數的海馬迴神經細胞上之rEag鉀離子通道分布狀況:rEag1和rEag2鉀離子通道都分布在神經細胞本體以及近端樹突,且rEag1鉀離子通道還分布到遠端樹突,並呈現斑點狀染色型態,顯示兩個鉀離子通道之次細胞層次分布的確不同。我們的溶解度分析實驗結果顯示rEag1和rEag2兩者對不同的清潔劑的溶解度有所不同:rEag1鉀離子通道在各種清潔劑中的溶解度相對高於rEag2鉀離子通道。進一步利用共同免疫沉澱的實驗,我們證實在HEK293T細胞株,rEag1與rEag2可能可以形成heterotetramer的鉀離子通道;而在大鼠大腦與海馬迴組織,rEag1與rEag2也可能有形成heterotetramer的鉀離子通道。但是進一步的分析發現有相當比例的rEag1是形成homotetramer的鉀離子通道。當我們以RT-PCR與real-time PCR偵測rEag1與rEag2 的mRNA於各腦區間的表現差異,顯示在大腦皮質處的rEag1與rEag2 mRNA含量遠高於其他腦區,且rEag1的含量高於rEag2。
綜合本研究的結果顯示rEag1與rEag2兩者在生化特性上有所差異,且彼此可形成heterotetramer或homotetramer的鉀離子通道。我們推測形成homotetramer和heterotetramer的rEag鉀離子通道的現象,可能是造成rEag1和rEag2在海馬迴神經細胞的次細胞分布不同的主要機制之一。
Functional voltage-gated potassium (K+) channel comprises the assembly of four pore-forming α-subunits in a homo- or hetero-tetrameric configuration. The ether-á-go-go (Eag) K+ channel is a member of the superfamily of voltage-gated K+ channel and broadly expressed over the entire central nervous system. There are two isoforms of Eag K+ channels in rat, rEag1 and rEag2. Previous studies from our lab have shown that rEag1 and rEag2 K+ channels display differential subcellular localization patterns in cultured hippocampal neurons. Both rEag1 and rEag2 K+ channels are expressed at soma and proximal dendrites; whereas only rEag1 channels display punctate staining patterns throughout the dendrites. The purpose of this study is to characterize the biochemical properties of rEag1 and rEag2 K+ channels. In particular, we investigated whether rEag1 and rEag2 subunits co-assemble to form heterotetrameric channels in rat forebrain and hippocampus, the result of which may shed light on the mechanism underlying the differential localization of rEag channels in neurons,
Firstly, we examined the immunoreactivities of rEag K+ channels in cultured hippocampal neurons that were subjected to different days in vitro. All neurons studied revealed a similar pattern of differential localization of rEag1 and rEag2 K+ channels. We then applied a variety of different detergents in rat brain homogenates to examine the relative solubility of rEag1 and rEag2 proteins. For the majority of the detergents used, the solubility of rEag1 subunits was significantly greater than that of rEag2. Furthermore, co-immunoprecipitation experiments showed that rEag1 and rEag2 K+ channels can be co-sedimented in cell lysates prepared from transfected HEK293T cells, forebrain and hippocampus, suggesting the formation of heterotetrameric K+ channels. Nevertheless, a considerable portion of rEag1 subunits were found to be homotetramers. Finally, RT-PCR and real-time PCR analyses of rEag1 and rEag2 mRNA in different brain regions suggested that the expression level of rEag1 subunit was significantly higher than that of rEag2 in most of the brain area.
In conclusion, our results indicate that rEag1 and rEag2 K+ channels exhibit different lipid-solubility, suggesting that they may be preferentially localized at distinct membrane microdomains. Moreover, in neurons rEag1 and rEag2 subunits may exist as either hetero- or homo-tetrameric K+ channels. We therefore propose that the differential tetrameric assembly configuration of rEag isomers may contribute to the subcellular localization pattern of rEag1 and rEag2 K+ channels.
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參考文獻…………………………………………………… 57
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