本文選題：神經(jīng)元鈣感應(yīng)蛋白(NCS) + KChIPs��； 參考：《復(fù)旦大學(xué)》2009年博士論文

【摘要】： 細(xì)胞內(nèi)鈣離子濃度對(duì)于維持正常的神經(jīng)元功能有著重要作用。研究表明,鈣離子的多種功能都必須通過(guò)鈣結(jié)合蛋白以及其與靶蛋白的互作來(lái)介導(dǎo),神經(jīng)元鈣感應(yīng)蛋白(Neuronal Calcium Sensors,NCS)家族是其中非常重要的一組。 神經(jīng)元鈣感應(yīng)蛋白家族屬于EF-hand類(lèi)Ca~(2+)結(jié)合蛋白超家族中的一個(gè)亞家族,在進(jìn)化過(guò)程中高度保守,典型的結(jié)構(gòu)特征是均含有3-4個(gè)EF-hand的Ca~(2+)結(jié)合功能域。迄今為止已有20多個(gè)不同物種的NCS蛋白被克隆,分屬3個(gè)亞類(lèi),第1類(lèi)包括visinin、recoverin和S-modulin;第2類(lèi)包括hippocalcin、frequenin和NCS-1;第3類(lèi)為4個(gè)新克隆的KChIP蛋白,KChIP1-4。KChIPs不同成員之間的相似性非常高,在氨基酸水平的相似性達(dá)到65%,其C端為3-4個(gè)EF-hand,N端的差異較大。 KChIPs能與A型鉀離子通道Kv4相互作用,發(fā)揮A型快鉀通道的β亞基的功能。KChIP1能減緩Kv4.2的失活時(shí)程,但卻顯著加速Kv4.1的失活。小鼠基因組中剔除KChIP2可導(dǎo)致心肌細(xì)胞Ca~(2+)依賴(lài)的短暫外向K~+離子電流完全丟失,KChIP2剔除小鼠表現(xiàn)為室性心動(dòng)過(guò)速,進(jìn)一步說(shuō)明了KChIPs有調(diào)節(jié)K~+離子通道的作用。KChIP3可以同Kv4.2、Kv4.3和Kv4.4通道相互作用,調(diào)節(jié)K+通道電流,從而在長(zhǎng)程增強(qiáng)和神經(jīng)元塑性中發(fā)揮重要的作用。KChIP4同樣具有同Kv4.2和Kv4.3通道相互作用的能力,突變KChIP4的Ca~(2+)結(jié)合模體可造成K+通道電導(dǎo)消失。 KChIPs在細(xì)胞內(nèi)還可以與多種蛋白發(fā)生相互作用,我們?cè)谶^(guò)去的研究中,用酵母雙雜交技術(shù)研究家族性大腸腺瘤息肉病致病基因APC(APC,Adenomatous polyposis coli)的中段互作蛋白過(guò)程中獨(dú)立克隆了KChIP1,提示KChIP1可能參與APC介導(dǎo)的神經(jīng)性調(diào)控。KChIP3可以同老年性癡呆(Alzheimer's Disease,AD)相關(guān)蛋白早老素presenilin結(jié)合;體外實(shí)驗(yàn)及KChIP3-/-小鼠體內(nèi)實(shí)驗(yàn)均證實(shí),其還可以與強(qiáng)啡肽反應(yīng)元件(DynorphinResponse Element,DRE)結(jié)合,抑制性調(diào)節(jié)Prodynorphin的表達(dá),從而調(diào)節(jié)痛覺(jué)反應(yīng),因此又被稱(chēng)為DREAM。此外,KChIP3還具有鈣依賴(lài)性的凋亡刺激作用和寡聚化作用。同樣,KChIP4也能與老年性癡呆相關(guān)基因早老素2(presenilin-2)相互作用,當(dāng)KChIP4單獨(dú)表達(dá)時(shí)呈胞漿和核彌散性分布,與presenilin-2共同表達(dá)時(shí)會(huì)發(fā)生核周和內(nèi)質(zhì)網(wǎng)上的共定位。 盡管已經(jīng)有了上述這些發(fā)現(xiàn),但KChIPs蛋白家族在其主要表達(dá)部位—腦的功能仍不清楚。本研究在前人的基礎(chǔ)上,對(duì)KChIP1基因在腦內(nèi)的功能進(jìn)行了深入地研究。整個(gè)研究分為3個(gè)部分。 第一部分通過(guò)組織原位雜交、免疫組化以及免疫染色研究,我們發(fā)現(xiàn)在成年小鼠腦中,KChIP1主要表達(dá)在一組小清蛋白(parvalbumin)陽(yáng)性的GABA(gamma amino butyric acid,γ-氨基丁酸,GABA)能神經(jīng)元,GABA傳導(dǎo)抑制性突觸傳遞,提示KChIP1可能在調(diào)節(jié)抑制性突觸傳遞中起作用。 第二部分;我們通過(guò)基因打靶、同源重組技術(shù)對(duì)KChIP1基因進(jìn)行了敲除,得到了KChIP1+/-和KChIP1-/-突變小鼠。雜合子和純合子小鼠在出生、發(fā)育等方面與野生型小鼠無(wú)顯著差異,并且符合孟德?tīng)栠z傳規(guī)律,提示KChIP1基因在小鼠胚胎發(fā)育中是非必須的,并且KChIP1基因?qū)τ谛∈蟪錾蟮纳L(zhǎng)發(fā)育也無(wú)顯著的影響。盡管如此,我們還是發(fā)現(xiàn)了一些有趣的現(xiàn)象:KChIP1+/-和KChIP1-/-小鼠做轉(zhuǎn)輪實(shí)驗(yàn)(Rotarod test)時(shí)較野生型小鼠更容易從轉(zhuǎn)輪中摔落下來(lái)(P＜0.05),提示其存在潛在的運(yùn)動(dòng)功能障礙;通過(guò)使用戊四氮(Pentylenetetrazole,PTZ)誘導(dǎo)癲癇發(fā)作,我們發(fā)現(xiàn)KChIP1+/-和KChIP1-/-小鼠較野生型小鼠更容易發(fā)生抽搐,并且致死率很高(P＜0.05),進(jìn)一步提示了KChIP1在調(diào)節(jié)抑制性突觸傳遞中的生理學(xué)作用。 第三部分中,我們?cè)贙ChIP1-/-突變小鼠的小腦組織中,克隆了一種新的KChIP1剪切體,我們命名為KChIP1c。先前的研究表明KChIP1具有兩個(gè)剪切變異體,稱(chēng)為KChIP1a和KChIP1b,后者N端包含另外一個(gè)外顯子。與KChIP1a和KChIP1b相似,在細(xì)胞水平,KChIP1c能與Kv4.3互作,并促進(jìn)Kv4.3的膜定位。在爪蟾的卵母細(xì)胞中,KChIP1c能增加Kv4.3電流的振幅,加速其在開(kāi)放狀態(tài)下的失活,促進(jìn)其在失活狀態(tài)下的快速恢復(fù)。然而,與KChIP1a和KChIP1b相比,KChIP1c比KChIP1a、KChIP1b更能增加Kv4.3電流的振幅,而且KChIP1c并不影響Kv4.3在關(guān)閉狀態(tài)下的失活。 綜上所述,通過(guò)上述研究,我們得出以下結(jié)論:KChIP1通過(guò)與細(xì)胞中Kv4.2和Kv4.3離子通道相互作用,引起細(xì)胞的興奮性發(fā)生改變,從而在調(diào)節(jié)機(jī)體運(yùn)動(dòng)平衡中發(fā)揮一定的作用;KChIP1通過(guò)與GABAergic神經(jīng)元細(xì)胞上的Kv4.2和Kv4.3通道相互作用,促進(jìn)GABA神經(jīng)遞質(zhì)的釋放,從而在對(duì)抗癲癇發(fā)作、維持神經(jīng)系統(tǒng)的興奮性狀態(tài)中發(fā)揮重要的作用;在腦內(nèi)存在著不同的KChIP1,其功能也不盡相同,在不同的部位對(duì)A型鉀離子通道起著不同的調(diào)節(jié)作用。以上結(jié)果為KChIP1相關(guān)退行性疾病診斷和治療研究奠定了良好的理論基礎(chǔ)。
[Abstract]:The intracellular calcium ion concentration plays an important role in maintaining normal neuronal function . The study shows that many functions of calcium ion must be mediated by calcium binding protein and its interaction with the target protein . The family of calcium sensing proteins ( NCS ) is a very important group .



There have been more than 20 different species of NCS protein which have been cloned , divided into 3 subfamilies . The species belong to three subfamilies . So far , there are more than 20 different species of NCS protein , including visinin , dimethyenin and NCS - 1 . The similarity between different members of KChIP protein , KChIP 1 - 4 and KChIPs in class 3 is very high , and the similarity of amino acid level reaches 65 % .



KChip1 can slow down the inactivation of KV4.2 , but significantly accelerate the inactivation of KV4.2 . In the mouse genome , KChIP can play an important role in regulating K ~ + ion channel . KChIP can play an important role in regulating K ~ + ion channel . KChIP 4 also has the ability to interact with Kv4.2 , K4.3 and Kv4.2 channels . The KChIP 4 has the same ability to interact with Kv4.2 and K4.3 channels .



In the past research , KChIP1 was isolated from the middle - segment interaction protein of familial colorectal adenomatous polyp disease ( APC ) by yeast two - hybrid technique , and KChIP 1 was also known as DREAM . In addition , KChIP 4 could interact with the presenilin - 2 gene .



Although the above findings have been found , the function of the KChIPs family in the main expression site - brain remains unclear . On the basis of the previous study , the function of the KChIP1 gene in the brain is studied in - depth . The whole study is divided into 3 parts .



In the first part , we found that , in adult mouse brain , KChIP1 was mainly expressed in a group of parvalrous positive GABA ( gamma amino butyric acid , 緯 - aminobutyric acid , GABA ) ergic neuron , GABA - conduction inhibitory synaptic transmission in adult mouse brain , suggesting that KChP 1 might play a role in regulating the inhibitory synaptic transmission .



The KChIP1 + / - and KChIP1 - / - mice were more likely to fall down from the rotating wheel ( P < 0.05 ) , and the KChIP1 + / - and KChIP1 - / - mice were more likely to have convulsions than wild - type mice , and the mortality was high ( P < 0.05 ) .



In the third part , we cloned a new KChIP1 shear body in the small brain tissue of KChIP1 - / - mutant mice , we named KChIP1c . The former research shows that KChIP1 has two shear variants , known as KChIP1a and KChIP1b . In the oocyte of Xenopus , KChIP1c can increase the amplitude of K4.3 current and accelerate its fast recovery in deactivated state . However , KChIP1c can increase the amplitude of K4.3 current in the open state than KChIP1a and KChIP1b . However , KChIP1c does not affect the deactivation of KV4.3 in the closed state .



In conclusion , through the above research , we conclude that KChIP1 plays an important role in regulating organism motion balance by interacting with Kv4.2 and K4.3 ion channels in the cells . The KChIP1 plays an important role in regulating the organism ' s motion balance . The KChIP1 plays an important role in regulating the excitatory state of the nervous system .
【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2009
【分類(lèi)號(hào)】：R346

【共引文獻(xiàn)】

相關(guān)期刊論文 前1條

1 于劍鋒;彭裕文;崔東紅;;APC基因與精神分裂癥及其他相關(guān)神經(jīng)精神疾病[J];中國(guó)神經(jīng)精神疾病雜志;2008年06期

相關(guān)博士學(xué)位論文 前1條

1 孟博;REGγ基因敲除小鼠類(lèi)精神分裂癥行為學(xué)及分子機(jī)制初探[D];華東師范大學(xué);2010年

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本文編號(hào)：1961711