钾离子通道互作蛋白-KChIP1基因的功能研究

发布时间：2018-05-31 23:08

本文选题：神经元钙感应蛋白(NCS) + KChIPs　；参考：《复旦大学》2009年博士论文

【摘要】： 细胞内钙离子浓度对于维持正常的神经元功能有着重要作用。研究表明,钙离子的多种功能都必须通过钙结合蛋白以及其与靶蛋白的互作来介导,神经元钙感应蛋白(Neuronal Calcium Sensors,NCS)家族是其中非常重要的一组。神经元钙感应蛋白家族属于EF-hand类Ca~(2+)结合蛋白超家族中的一个亚家族,在进化过程中高度保守,典型的结构特征是均含有3-4个EF-hand的Ca~(2+)结合功能域。迄今为止已有20多个不同物种的NCS蛋白被克隆,分属3个亚类,第1类包括visinin、recoverin和S-modulin;第2类包括hippocalcin、frequenin和NCS-1;第3类为4个新克隆的KChIP蛋白,KChIP1-4。KChIPs不同成员之间的相似性非常高,在氨基酸水平的相似性达到65%,其C端为3-4个EF-hand,N端的差异较大。 KChIPs能与A型钾离子通道Kv4相互作用,发挥A型快钾通道的β亚基的功能。KChIP1能减缓Kv4.2的失活时程,但却显著加速Kv4.1的失活。小鼠基因组中剔除KChIP2可导致心肌细胞Ca~(2+)依赖的短暂外向K~+离子电流完全丢失,KChIP2剔除小鼠表现为室性心动过速,进一步说明了KChIPs有调节K~+离子通道的作用。KChIP3可以同Kv4.2、Kv4.3和Kv4.4通道相互作用,调节K+通道电流,从而在长程增强和神经元塑性中发挥重要的作用。KChIP4同样具有同Kv4.2和Kv4.3通道相互作用的能力,突变KChIP4的Ca~(2+)结合模体可造成K+通道电导消失。 KChIPs在细胞内还可以与多种蛋白发生相互作用,我们在过去的研究中,用酵母双杂交技术研究家族性大肠腺瘤息肉病致病基因APC(APC,Adenomatous polyposis coli)的中段互作蛋白过程中独立克隆了KChIP1,提示KChIP1可能参与APC介导的神经性调控。KChIP3可以同老年性痴呆(Alzheimer's Disease,AD)相关蛋白早老素presenilin结合;体外实验及KChIP3-/-小鼠体内实验均证实,其还可以与强啡肽反应元件(DynorphinResponse Element,DRE)结合,抑制性调节Prodynorphin的表达,从而调节痛觉反应,因此又被称为DREAM。此外,KChIP3还具有钙依赖性的凋亡刺激作用和寡聚化作用。同样,KChIP4也能与老年性痴呆相关基因早老素2(presenilin-2)相互作用,当KChIP4单独表达时呈胞浆和核弥散性分布,与presenilin-2共同表达时会发生核周和内质网上的共定位。尽管已经有了上述这些发现,但KChIPs蛋白家族在其主要表达部位—脑的功能仍不清楚。本研究在前人的基础上,对KChIP1基因在脑内的功能进行了深入地研究。整个研究分为3个部分。第一部分通过组织原位杂交、免疫组化以及免疫染色研究,我们发现在成年小鼠脑中,KChIP1主要表达在一组小清蛋白(parvalbumin)阳性的GABA(gamma amino butyric acid,γ-氨基丁酸,GABA)能神经元,GABA传导抑制性突触传递,提示KChIP1可能在调节抑制性突触传递中起作用。第二部分;我们通过基因打靶、同源重组技术对KChIP1基因进行了敲除,得到了KChIP1+/-和KChIP1-/-突变小鼠。杂合子和纯合子小鼠在出生、发育等方面与野生型小鼠无显著差异,并且符合孟德尔遗传规律,提示KChIP1基因在小鼠胚胎发育中是非必须的,并且KChIP1基因对于小鼠出生后的生长发育也无显著的影响。尽管如此,我们还是发现了一些有趣的现象:KChIP1+/-和KChIP1-/-小鼠做转轮实验(Rotarod test)时较野生型小鼠更容易从转轮中摔落下来(P＜0.05),提示其存在潜在的运动功能障碍;通过使用戊四氮(Pentylenetetrazole,PTZ)诱导癫痫发作,我们发现KChIP1+/-和KChIP1-/-小鼠较野生型小鼠更容易发生抽搐,并且致死率很高(P＜0.05),进一步提示了KChIP1在调节抑制性突触传递中的生理学作用。第三部分中,我们在KChIP1-/-突变小鼠的小脑组织中,克隆了一种新的KChIP1剪切体,我们命名为KChIP1c。先前的研究表明KChIP1具有两个剪切变异体,称为KChIP1a和KChIP1b,后者N端包含另外一个外显子。与KChIP1a和KChIP1b相似,在细胞水平,KChIP1c能与Kv4.3互作,并促进Kv4.3的膜定位。在爪蟾的卵母细胞中,KChIP1c能增加Kv4.3电流的振幅,加速其在开放状态下的失活,促进其在失活状态下的快速恢复。然而,与KChIP1a和KChIP1b相比,KChIP1c比KChIP1a、KChIP1b更能增加Kv4.3电流的振幅,而且KChIP1c并不影响Kv4.3在关闭状态下的失活。综上所述,通过上述研究,我们得出以下结论:KChIP1通过与细胞中Kv4.2和Kv4.3离子通道相互作用,引起细胞的兴奋性发生改变,从而在调节机体运动平衡中发挥一定的作用;KChIP1通过与GABAergic神经元细胞上的Kv4.2和Kv4.3通道相互作用,促进GABA神经递质的释放,从而在对抗癫痫发作、维持神经系统的兴奋性状态中发挥重要的作用;在脑内存在着不同的KChIP1,其功能也不尽相同,在不同的部位对A型钾离子通道起着不同的调节作用。以上结果为KChIP1相关退行性疾病诊断和治疗研究奠定了良好的理论基础。
[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 , 纬 - 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 .
【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R346

【共引文献】