Title

大鼠Eag鉀離子通道次細胞分佈差異之結構性基礎

Translated Titles

Structural Basis of the Differential Subcellular Localization of Rat Eag K+ Channels

DOI

10.6342/NTU.2006.02036

Authors

林惠敏

Key Words

Eag ; 鉀離子通道 ; Eag ; potassium channel

PublicationName

臺灣大學生理學研究所學位論文

Volume or Term/Year and Month of Publication

2006年

Academic Degree Category

碩士

Advisor

湯志永

Content Language

繁體中文

Chinese Abstract

EAG鉀離子通道屬於電位控制鉀離子通道大家族之一,而根據胺基酸序列之相異性,又可細分為eag、erg、elk三種次家族。在哺乳動物中eag次家族成員包含Eag1以及Eag2鉀離子通道,此二離子通道主要分佈於中樞神經系統中。本實驗室先前針對大鼠Eag1(rEag1)與Eag2(rEag2)在海馬迴神經細胞中之次細胞分佈情形的研究結果顯示,rEag1主要分佈於細胞本體、近端樹突、及遠端樹突;而rEag2則主要分佈於細胞本體及近端樹突。此外rEag1在樹突上具有明顯的點狀(cluster)分佈,由於這些點狀分部與突觸標地(marker)具有colocalization之現象,顯示rEag1鉀離子通道還分佈於神經突觸之位置。 本篇論文之目的為探討rEag1與rEag2此二離子通道在神經細胞中的次細胞分佈狀況不同的分子機制為何。由胺基酸序列可知,rEag1與rEag2在core region之extracellular loops中,所包含之N-linked glycosylation sites有所不同,其中rEag1在胺基酸序列中388及406兩個位置具有glycosylation sites,而在rEag2中此相對位置不具glycosylation consensus sequence。另外,rEag1與rEag2之胺基酸序列,其最大差異處在於C端。因此綜合以上,我們首先要驗證之假設為rEag1在胺基酸序列388或406 glycosylation pattern可能與rEag1與rEag2在神經細胞之次細胞分佈之差異有關。第二,我們也將驗證決定rEag1與rEag2之targeting的差異亦可能在於C端。再者,內源性鉀離子通道中,同一次家族成員彼此間可能形成hetero-tetramer,過去研究證實rEag1與rEag2在大鼠中樞神經系統中之分佈確實具有部分重疊性,因此我們將驗證之假設三為,若rEag1與rEag2在in vivo當中能夠彼此聚合,當rEag1與rEag2 subunits共同形成四聚體時,能由某方主導次細胞之分佈位置。 在實驗方式上,我們利用GFP tagged之rEag1(GFP-rEag1)於388、406此二位置作點突變,使之廢除glycosylation sites,再將此二mutant transfect到培養之神經細胞當中,利用confocal image觀察其在神經細胞中之subcellular localization。接者為證實是否為C端決定targeting之所在,我們將GFP-rEag1與GFP-rEag2之C端序列作交換,使產生reag2-chimera 1、reag2-chimera 2、reag1-chimera 1之結構,亦將其transfect到培養之神經細胞當中,觀察其表現模式,結果證實rEag1與rEag2 C端之差異,確實可能為造成兩者在神經細胞內差異的主要因素。再者為驗證rEag1與rEag2在in vivo中彼此聚合之可能性,我們將rEag1與GFP-rEag2共同在HEK293T細胞中表達,以及將腦組織研磨後純化出細胞膜蛋白後,再以免疫沉澱的方式作為佐證依據,結果顯示,在HEK293T以及鼠腦組織膜蛋白萃取物中,皆顯示出rEag1與rEag2能夠相互結合,證實兩者在in vivo中形成heterotetramer之可能性。 綜合以上,我們證實rEag channels之C端為影響神經細胞內次細胞分佈之重要因子,且rEag1與rEag2在in vivo當中確實能彼此聚合,形成hetero-tetramer之離子通道結構。

English Abstract

The EAG potassium channel is a member of the superfamility of voltage- gated potassium channels, and can be subdivided into three different subfamilies based on sequence homology:eag, erg and elk. In mammalians, the eag subfamily includes Eag1 and Eag2 K channels, which have been shown to express exclusively in central nervous system. Previous studies from our lab have demonstrated that rat Eag1 and rat Eag2 displayed differential subcellular localization patterns in hippocampus neurons. In the present study, we aim to determine the structural basis of differential localization of rEag1 and rEag2 in neurons. We intend to test the following hypotheses:(1) the differential localization pattern may be related to the different glycosylation patterns between rEag1 and rEag2. (2) the intracellular carboxyl terminal(C-terminal) region of rEag1 and rEag2 may contain the targeting signal that dictates the subcellular localization patterns of the two channels, and (3) rEag1 and rEag2 may form hetero-tetramers in neurons, suggesting the existence of a dominancy in the subcellular targeting within hetero-tetramer. We first studied the effect of abolishing either of the two known glycosylation sites in GFP-rEag1. Our results did not suppory a role of glycosylation in the subcellular localization of rEag1. We next engineered several chimeras that involved the C terminal regions of rEag1 and rEag2. By studing the confocal image of neuron transfected chimeric GFP constructs, we found evidence showing the C –terminal regions of rEag channels may confer the differential localization patterns of the two channels. Finally, we performed biochemical experiments to confirm that rEag1 and rEag2 are capable of forming hetero-tetramers in vivo. In conclusion, the C terminals of rEag channels play an important role in differential localization in neurons and rEag1 and rEag2 can assemble with each other in vivo.

Topic Category 醫藥衛生 > 基礎醫學
醫學院 > 生理學研究所
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