rEag1鉀離子通道訊息與生合成之分子調控機制

Molecular regulation of rEag1 K+ channel signaling and biosynthesis

許博皓(Po-Hao Hsu)

指導教授 ： 湯志永

國立臺灣大學/醫學院/生理學研究所/博士(2015年)

https://doi.org/10.6342/NTU.2015.11107

摘要

在神經系統中，電位調控鉀離子通道對於維持正常神經生理功能扮演重要的角色，包括維持膜電位、媒介細胞內的分子訊息傳導路徑、調控細胞週期等。Eag1鉀離子通道屬於ether-à-go-go (EAG)鉀離子通道家族，屬於電位調控之鉀離子通道。在哺乳動物中，Eag鉀離子通道表現於神經組織且廣泛分佈於許多不同腦區，但目前對其詳細生理功能仍不清楚。我們利用酵母菌雙雜交系統篩選與大鼠Eag1 (rEag1)有相互作用之蛋白質。其中三個可能與rEag1具相互作用的蛋白質為14-3-3θ、centrin 4、cullin 7。在本篇研究中，我們在in vivo與in vitro確認rEag1與其結合蛋白間的相互作用關係，並進一步探討這些與rEag1有相互作用的蛋白質如何調控rEag1鉀離子通道的功能與生合成機制。
14-3-3θ屬於小型酸性蛋白家族成員之一並大量表現於大腦中。目前已知14-3-3蛋白質能與超過一百種蛋白質相互作用，參與調控許多細胞內的分子機制。首先我們利用共同免疫沉澱與GST pull-down實驗確認14-3-3θ與rEag1之相互作用關係，並且利用免疫細胞染色與次細胞分布離心分離實驗進行驗證此二蛋白質間是否在空間上分佈於相同區域。電生理實驗中，顯示大量表現14-3-3θ蛋白時，rEag1鉀離子通道電流有顯著下降的情形，此電流減少並不影響rEag1單一離子通道之電生理特性，也不影響蛋白質生成與細胞膜上蛋白表現量。因此推論14-3-3θ的作用可能是使rEag1鉀離子通道蛋白質構型在不導電狀態，而導致電流下降。
Centrin 4為具有鈣離子結合位之centrin家族一員，在哺乳動物細胞中主要表現於中心體相關結構並參與調控細胞週期。在視網膜中，centrin表現在視網膜光細胞的中心體與基體的結構，參與transducin的運送。我們利用共同免疫沉澱驗證centrin 4與rEag1之相互作用關係，並進一步藉由GST pull-down推論rEag1主要是藉由位於CNBHD之後約100個胺基酸大小的區域與centrin 4進行結合。另一方面，我們證實鈣離子存在與否並不影響此相互作用關係。
Cullin 7為E3泛素連接酶 (ubiquitin ligase)， 屬於culllin-RING家族成員之一。我們利用共同免疫沉澱與GST pull-down實驗確認cullin 7與rEag1之相互作用。cullin 7的大量表現，會提升rEag1的泛素化修飾作用，並縮短其半衰期，而降低rEag1表現量。另一方面，當以siRNA或shRNA剔除cullin 7表現時，rEag1表現量則有顯著增加。當以20 μM NMDA誘導神經毒性去處理培養的大腦皮質神經細胞時，造成rEag1表現明顯降低。當以蛋白體酶抑制劑預處理神經細胞，則能避免神經毒性導致的rEag1表現量減少，因此我們推測cullin 7可能參與在NMDA造成rEag1表現減少的降解機制中。

關鍵字

rEag1鉀離子通道 ； 14-3-3θ蛋白質 ； centrin 4蛋白 ； cullin 7 E3泛素連接? ； 蛋白質相互作用

並列摘要

Voltage-gated potassium (K+) channels contribute to many neurophysiological functions in th nervous system, including setting the membrane potential, modulating cell signaling transduction, and controlling cell cycle progression.The Eag1 K+ channel is a member of the ether-à-go-go (EAG) K+ channel family that belong to the superfamily of voltage-gated K+ channels. In mammals, the expression of Eag1 K+ channel is neuron-specific and broadly expressed in the various region of the central nervous system, but the physiological functions of Eag1 K+ channel are still unclear. We performed the yeast two-hybrid screening to identify the rat Eag1 (rEag1) interacting proteins. Three clones, 14-3-3θ, centrin 4 and cullin 7, were identified. In this study, I have confirmed the interaction of rEag1-interacting proteins with rEag1 both in vivo and in vitro. In addition, I have investigated how rEag1-interacting proteins affect the biosynthesis and functional properties of rEag1 channels.
14-3-3θ belongs to a family of small acidic protein abundantly expressed in the brain. The 14-3-3 proteins have been shown to interact with more than one hundred of proteins and may mediate diverse regulatory functions in many cellular processes. We have performed the co-immunoprecipitation and GST pull-down assay to confirm the interaction between 14-3-3θ and rEag1. To further address if rEag1 and 14-3-3θ shared overlapping subcellular localization, we applied immunofluorescence staining and subcellular fractionation experiment to compare the subcellular distribution. Electrophysiological studies showed that over-expression of 14-3-3θ led to suppression of rEag1 K+ currents without alteration of the gating kinetics and single channel conductance of rEag1. Co-expression with 14-3-3θ also failed to affect the total protein level and memebrane trafficking. These were implied 14-3-3θ binding may block the rEag1 K+ channel in a non-conducting state.
Centrin 4 is a member of the centrin protein family, which is the parvalbumin superfamily of Ca2+ binding proteins. Centrins are expressed and associated with centrosome-related structures in mammals. In previous studies on the retina have show that centrins are the components of centrosomes and basal bodies in photoreceptors, and involved in the transducin transport and supply. I have confirmed the interaction between rEag1 and centrin 4 by GST pull-down and co-immunoprecipitation. GST pull-down assay indicated that centrin 4 bound to the motif after CNBHD domain of rEag1. In addition, I found that calcium was not necessary for the interaction between rEag1 and centrin 4.
Cullin 7 belongs to the cullin RING-finger E3 ligases which represent the largest E3 ubiquitin ligase family in eukaryotes. I have performed the co-immunoprecipitation and GST pull-down assay to confirm the interaction between cullin 7 and rEag1 in heterologous cells and neuronal tissues. Overexpression of cullin 7 decreased rEag1 expression by increasing the ubiquitinylation and shortening the half-life of rEag1. In contrast, knockdown of cullin 7 by either siRNA or shRNA dramatically increased the expression of rEag1. In cultured cortical neurons, following 20 μM NMDA treatment to induce excitotoxicity, rEag1 protein level was dramatically drcreased. This NMDA-induced reduction of rEag1 protein was prevented by pretreatment with proteasome inhibitors, MG132 and ALLN. It is possible that cullin 7 is responsible for the NMDA-mediated reduction of rEag1 proteins in neurons.

並列關鍵字

rEag1 potassium channel ； 14-3-3θ ； centrin 4 ； cullin 7 E3 ligase ； protein-protein interaction

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