利用酵母雙雜交技術找尋在大鼠腦中與P/Q-型鈣離子通道有交互作用的蛋白質

Abstract

P/Q-型鈣離子通道是由形成離子孔道 (pore-forming)的α1A subunit與其他α2δ、β等auxiliary subunit組成。絕大多數的P/Q-型鈣離子通道是位於神經細胞的樹突和軸突末端，因此其神經生理功能主要是神經細胞的興奮性及調控突觸神經傳導物質的作用。P/Q-型鈣離子通道之突變與多種遺傳性神經病變有關，詳細機制則尚未完全清楚。目前已知P/Q-型鈣離子通道調控蛋白質包括：SNARE proteins、G proteins、calmodulin (CaM)、RIM (Rab3-interacting molecules)及RIMBP (RIM binding protein)等，多數是經由離子通道的C-terminus產生作用。為了進一步尋找在神經細胞中其他可能和C-terminus結合的蛋白質，我們以酵母雙雜交技術篩檢在大鼠腦中與P/Q-型鈣離子通道交互作用的蛋白質。 對大鼠腦部cDNA library進行篩選後，我們共發現了156個蛋白質可能與P/Q-型鈣離子通道有交互作用。在經過進一步的序列分析和扣除胺基酸序列有frame shift的選項後，剩下67個可能P/Q-型鈣離子通道有相互作用的蛋白質。我們從中挑選10個蛋白質，以進一步的實驗以確認其與P/Q-型鈣離子通道的相互作用能力。X-gal測試分析和白胺酸需求分析實驗的結果顯示，這10個蛋白質都能在進行X-gal分析時使菌落由白變藍，並且在缺乏白胺酸的培養皿上生長。我們進一步以GST pull-down之方式驗證，發現其中的7個得到正反應。另外，在Co-IP檢驗中，目前檢驗了其中3個，而在之中的2個蛋白質有正反應。接著，我們以免疫螢光染色的實驗方法，由共軛焦顯微鏡影像觀察與P/Q-型鈣離子通道交互作用的蛋白質，在細胞中的分布情形。藉此實驗我們可得知有哪些蛋白質和P/Q-型鈣離子通道分布位置重合。 針對本篇研究所篩選到的蛋白質，我們除了以不同的生化實驗證明它們的交互作用外，我們期望藉此由後續的電生理實驗方式，證明對於P/Q-型鈣離子通道性質的影響，而能對於P/Q-型鈣離子通道之生理機制以及相關遺傳性神經退化性疾病，有更進一步的了解。

P/Q type voltage-gated Ca2+ channels are composed of pore-forming α1A subunit and auxiliary α2δ and β subunits. In neurons, P/Q type voltage-gated Ca2+ channels are primarily located at dendrite and axon terminal, the neurophysiological role of P/Q type voltage-gated Ca2+ channels may include the neuronal excitability and modulation of synaptic transmission. Mutations in P/Q type voltage-gated Ca2+ channels are associated with a variety of hereditary neuropathy, the detail mechanisms of which remain unclear. P/Q type voltage-gated Ca2+ channels were regulated by SNARE proteins, G proteins and calmodulin (CaM). In this study, we aim to apply the cytoplasma C-terminus of P/Q type voltage-gated Ca2+ channels as the bait for yeast two-hybrid screening to search for novel P/Q type voltage-gated Ca2+ channels interacting proteins. 156 prey clones were identified after screening a rat brain cDNA library. By DNA sequencing and eliminating the clones with incorrect reading frames, we have obtained 67 positive clones. 10 potential candidates from 67 positive clones were chosen for further characterization. X-gal assays and leucine requirement tests were performed to reconfirm the interaction between P/Q type voltage-gated Ca2+ channels and potential candidate proteins. All of the 10 candidate proteins showed blue patches on blue and/white tests and were able to grow on leucine-deficient plates, suggesting that these clones may indeed interact with P/Q type voltage-gated Ca2+ channels. We performed GST pull-down assay, and found the 7 of the 10 candidate proteins have positive reaction. We performed Co-IP assay, so far, we test 3 of these candidate proteins and found the 2 of the 3 candidate proteins have positive reaction. And then, we performed immunofluorescence microscopy imaging to observe subcelluar lococaliztion of these candidate proteins. We can find which candidate protein might colocolize with P/Q-type calcium channels. We expect to take electrophysiological experiments prove P/Q-type calcium channel properties, in addition to prove their interactions with different biochemical experiments. We hope to better understand about physiological mechanism of the P/Q-type calcium channels and the related hereditary neurodegenerative disease.