本文選題：NMDA受體 + 內(nèi)質(zhì)網(wǎng)滯留基序�。� 參考：《浙江大學(xué)》2008年博士論文

【摘要】： NMDA受體是一種谷氨酸門控的離子通道,與許多復(fù)雜的生理和病理機(jī)制有關(guān),如突觸的可塑性、長(zhǎng)時(shí)程增強(qiáng)作用(LTP)、學(xué)習(xí)和記憶、興奮性神經(jīng)毒性、神經(jīng)退行性變性疾病等。NMDA受體由NR1、NR2和NR3亞單位裝配組成異聚體離子通道,在內(nèi)質(zhì)網(wǎng)組成具有不同生理學(xué)和藥理學(xué)功能以及不同突觸靶向的功能性NMDA受體通道。已知NR1亞單位是一個(gè)獨(dú)立的基因家族,通過選擇性剪接可產(chǎn)生8個(gè)剪接變體;NR2共有4個(gè)相關(guān)的基因,NR2A、NR2B、NR2C和NR2D:NR3則有2個(gè)基因,分別為NR3A和NR3B。目前認(rèn)為,NMDA受體是分別由兩個(gè)結(jié)合甘氨酸的NR1和兩個(gè)結(jié)合谷氨酸的NR2亞單位組成的異四聚體。因此,NR1/NR2異源二聚體就組成了NMDA受體的基本功能單位。先前的大量研究表明,NMDA受體的不同亞單位單獨(dú)表達(dá)都被滯留在內(nèi)質(zhì)網(wǎng)內(nèi),只有當(dāng)NR1與NR2亞單位共同裝配才能從內(nèi)質(zhì)網(wǎng)輸出并表達(dá)到細(xì)胞膜表面,但其作用機(jī)制仍不清楚。近幾年來,研究發(fā)現(xiàn)NR1亞單位存在一些重要的功能區(qū)和調(diào)節(jié)位點(diǎn):例如,NR1-1亞單位的C1區(qū)存在一個(gè)RRR內(nèi)質(zhì)網(wǎng)滯留基序(ERretention motif),使NR1亞單位被滯留在內(nèi)質(zhì)網(wǎng);NR1-4a存在一個(gè)共感的PDZ結(jié)合區(qū),可以掩蓋內(nèi)質(zhì)網(wǎng)滯留基序;PKC活化可以幫助NR1-1a表達(dá)于細(xì)胞膜表面等等。然而,對(duì)于NR2B亞單位的內(nèi)質(zhì)網(wǎng)滯留機(jī)制卻仍然知之甚少。與NR1不同的是NR2B亞單位的C末端很長(zhǎng),我們先前的研究已經(jīng)發(fā)現(xiàn),缺失NR2B亞單位C末端不同區(qū)域均未能使得NR2亞單位從內(nèi)質(zhì)網(wǎng)輸出并獲得表面表達(dá),這表明NR2亞單位的內(nèi)質(zhì)網(wǎng)滯留機(jī)制要比NR1復(fù)雜得多。已知NR1-4a單獨(dú)表達(dá)能到達(dá)細(xì)胞膜表面,為了明確是否是NR2B亞單位C末端決定了它的自身滯留,我們將NR1-4a的C末端替換到NR2B的亞單位C末端。有趣的是,我們發(fā)現(xiàn)這樣的一個(gè)NR2B_(NR4aC)的嵌合體獲得了細(xì)胞膜表面表達(dá),這個(gè)結(jié)果表明NR2B亞單位C末端決定了NR2B亞單位的細(xì)胞膜表達(dá)。但是關(guān)于NR2B亞單位C末端如何調(diào)控NR2B亞單位的內(nèi)質(zhì)網(wǎng)滯留作用仍然不清楚,我們推測(cè)并不是由單一位點(diǎn)決定,很可能是通過多個(gè)位點(diǎn)協(xié)同作用的結(jié)果,這還需要進(jìn)一步的研究證明。另外,關(guān)于NR2亞單位單獨(dú)表達(dá)是否能形成功能性受體的研究一直是這個(gè)領(lǐng)域的一個(gè)熱點(diǎn),目前的主流觀點(diǎn)推測(cè)NR1是NMDA受體得必需組分,NR2亞單位單獨(dú)并不能形成有功能得離子通道�；贜R2B_(NR4ac)能單獨(dú)到達(dá)細(xì)胞膜表面,我們運(yùn)用全細(xì)胞電生理的方法檢測(cè)了NR2B_(NR4ac)嵌合體是否能形成有功能的受體通道。電生理結(jié)果沒有記錄到明確的NMDA受體樣電流。這個(gè)結(jié)果提示單獨(dú)表達(dá)NR2亞單位并不能形成有功能的受體通道,只有與NR1共裝配才能形成有功能的通道。 其次,已知NR1-1a可以通過與NR2裝配克服其內(nèi)質(zhì)網(wǎng)滯留作用并被輸送到細(xì)胞膜表面,但其作用機(jī)制并不清楚。我們先前的研究試圖在NR2上找到克服NR1a內(nèi)質(zhì)網(wǎng)滯留基序的功能區(qū),做了各種缺失突變體,但均未能找到特殊的功能區(qū)。在本研究中,我們構(gòu)建了NR2B各種C末端截短質(zhì)粒,結(jié)果發(fā)現(xiàn)除了2BA2不能與NR1-1a獲得細(xì)胞膜表面表達(dá)之外,其它的缺失體都能與NR1-1a共表達(dá)于細(xì)胞膜表面。進(jìn)一步用全細(xì)胞膜片鉗技術(shù)來記錄轉(zhuǎn)染細(xì)胞的NMDA受體電流,結(jié)果與表面染色結(jié)果一致。這些結(jié)果表明,NR2B的C末端氨基酸長(zhǎng)度對(duì)克服NR1-1a的內(nèi)質(zhì)網(wǎng)滯留有重要作用,換句話說,NR2亞單位C末端保留三個(gè)氨基酸長(zhǎng)度對(duì)于克服NR1-1a的內(nèi)質(zhì)網(wǎng)滯留基序作用是必需的。為了驗(yàn)證這個(gè)結(jié)果,我們獲得了一個(gè)NR1-1a-AAA突變體,只把NR1-1a的RRR滯留基序突變成了AAA。然后分別把2B△2與NR1-1a-AAA突變體共轉(zhuǎn)染到HEK293細(xì)胞,表面染色結(jié)果顯示它們能獲得細(xì)胞膜表達(dá)。接下來我們想知道的一個(gè)問題是這個(gè)氨基酸序列是否具有特異性呢?為了回答這個(gè)問題,我們構(gòu)建了NR2B△5_(AAAAA)突變體,將它與NR1-1a共轉(zhuǎn)染到HEK293細(xì)胞后,表面染色和電生理結(jié)果都表明它們形成的復(fù)合物仍然能夠到達(dá)細(xì)胞膜表面并形成有功能的受體。這個(gè)結(jié)果表明NR2B第四跨膜區(qū)后三個(gè)氨基酸長(zhǎng)度并沒有序列特異性�；谏鲜鼋Y(jié)果,本研究第一次發(fā)現(xiàn)NR2亞單位C末端保留三個(gè)氨基酸長(zhǎng)度對(duì)于克服NR1-1a的內(nèi)質(zhì)網(wǎng)滯留基序是必需的。這種調(diào)控機(jī)制很可能是通過引發(fā)NR1亞單位C末端自身變構(gòu)的改變,從而掩蓋了NR1-1a C末端的內(nèi)質(zhì)網(wǎng)滯留基序,使得NR2△3/NR1-1a獲得細(xì)胞膜表面表達(dá)。本研究揭示了一種新的NMDA受體亞單位裝配和運(yùn)輸調(diào)控機(jī)制。 另外,本研究運(yùn)用FRET技術(shù)來觀察NR2B亞單位的C末端是否影響NMDA受體的裝配。將構(gòu)建好的YFP-2B△2,CFP-2B△2質(zhì)粒共轉(zhuǎn)染到HEK293細(xì)胞,發(fā)現(xiàn)它們?nèi)匀荒軌虬l(fā)生FRET,這表明YFP-2B△2與CFP-2B△2能形成復(fù)合物。同時(shí),分別將YFP-2B△2與CFP-NR2B或者CFP-NR1-1a共轉(zhuǎn)染到HEK 293細(xì)胞,結(jié)果發(fā)現(xiàn)它們也能發(fā)生FRET。這些結(jié)果說明缺失了C末端的NR2B亞單位仍然能夠與NR1裝配形成NMDA受體復(fù)合物,提示NR2B的C末端并不是NMDA受體裝配的決定位點(diǎn)。 最后,在培養(yǎng)的海馬神經(jīng)元中分別單獨(dú)轉(zhuǎn)染GFP-NR2A,GFP-NR2B,2A△5,2A△3,2A△2,2B△3和2B△2,活細(xì)胞表面染色觀察它們?cè)谏窠?jīng)元的細(xì)胞膜表達(dá)情況。結(jié)果顯示2A△5,2A△3,2A△2單獨(dú)表達(dá)到神經(jīng)元均能到達(dá)細(xì)胞膜表面,盡管與野生型相比有顯著降低。有趣的是,2B△2單獨(dú)表達(dá)在神經(jīng)元中并不能獲得表面表達(dá),但是將NR1-4a與2B△2共轉(zhuǎn)染到神經(jīng)元中則能夠觀察到2B△2到達(dá)了細(xì)胞膜表面,雖然跟野生型相比膜表面表達(dá)數(shù)目仍然有明顯下降。眾所周知,在神經(jīng)元中存在各種內(nèi)源性的NR1剪接變體,如果NR2能與這些不同的NR1變體裝配的話,那么2B△2應(yīng)該能與NR1-4亞單位裝配并到達(dá)細(xì)胞膜表面,但結(jié)果發(fā)現(xiàn)2B△2不能到達(dá)細(xì)胞膜表面。這個(gè)結(jié)果暗示了一種新的現(xiàn)象,即不同的NR2亞單位并不是隨機(jī)的與各種NR1變體裝配形成NMDA受體,很可能與NR2A和NR2B裝配的NR1是不同的變體。 綜上,本研究主要結(jié)論如下:1)發(fā)現(xiàn)NR2B_(NR4aC)的嵌合體能獲得細(xì)胞膜表面表達(dá),電生理結(jié)果提示單獨(dú)表達(dá)NR2亞單位不能形成功能性離子通道;2)本研究首次發(fā)現(xiàn)NR2亞單位C末端保留三個(gè)氨基酸長(zhǎng)度對(duì)于克服NR1-1a的內(nèi)質(zhì)網(wǎng)滯留基序是必需的,揭示了一種新的NMDA受體亞單位裝配和運(yùn)輸調(diào)控機(jī)制。
[Abstract]:NMDA receptor is a glutamate gated ion channel, which is related to many complex physiological and pathological mechanisms, such as synaptic plasticity, long term enhancement (LTP), learning and memory, excitatory neurotoxicity, neurodegenerative disease, etc..NMDA receptors are assembled by NR1, NR2 and NR3 subunits to form an isomer ion channel in the endoplasmic reticulum group. As a functional NMDA receptor channel with different physiological and pharmacological functions and different synaptic targets, the known NR1 subunit is an independent gene family, and 8 splice variants can be produced by selective splicing; NR2 has 4 related genes, NR2A, NR2B, NR2C and NR2D:NR3 have 2 genes, NR3A and NR3B. are currently considered, NMD, respectively, NMD. The A receptor is an hetero four polymer consisting of two binding glycine NR1 and two NR2 subunits that bind glutamate. Therefore, NR1/NR2 heterologous two polymers constitute the basic functional units of the NMDA receptor. A large number of previous studies showed that the different subunits of the NMDA receptor were isolated in the endoplasmic reticulum alone, only when NR1 and NR2 subunits were subdivided. Unit assembly can be assembled from the endoplasmic reticulum to the surface of the cell membrane, but the mechanism of its action is still unclear. In recent years, some important functional areas and regulatory sites have been found in NR1 subunits, for example, a RRR endoplasmic reticulum retention base (ERretention motif) exists in the C1 region of the subunit of NR1-1 and the NR1 subunit is detained. NR1-4a has a shared PDZ binding area that can cover the retention of the endoplasmic reticulum; PKC activation can help NR1-1a to express on the surface of the cell membrane. However, little is known about the mechanism of the endoplasmic reticulum retention in NR2B subunits. Unlike NR1, the C end of the NR2B subunit is long, and our previous study has found the deletion. The C terminal of the subunit of the NR2B subunit failed to make the NR2 subunit output from the endoplasmic reticulum and obtain the surface expression. This indicates that the endoplasmic reticulum retention mechanism of the NR2 subunit is much more complex than that of NR1. The known NR1-4a expression can reach the surface of the cell membrane. In order to determine whether it is the C terminal of the NR2B subunit, it determines its self retention. We will NR1-4a The C terminal was replaced by the subunit C terminal of NR2B. Interestingly, we found that such a NR2B_ (NR4aC) chimeras obtained the expression of the cell membrane surface. This result indicates that the C terminal of the NR2B subunit determines the cell membrane expression of the subunit of NR2B, but how the end of the C end of NR2B subunit regulates the retention of the endoplasmic reticulum of NR2B subunit. It is still not clear that we speculate that it is not determined by a single locus, it is likely to be the result of synergistic effects of multiple sites, and this needs further research. In addition, the study of whether NR2 subunits can form functional receptors alone has been a hot spot in this field, and the current mainstream view is that NR1 is NMDA. The receptor is essential component, NR2 subunit alone does not form a functional ion channel. Based on the ability of NR2B_ (NR4ac) to reach the surface of the cell membrane alone, we use the whole cell electrophysiological method to detect whether the NR2B_ (NR4ac) chimeras can form a functional receptor channel. Electrophysiological results do not record a clear NMDA receptor like current. This result suggests that NR2 subunits alone can not form functional receptor channels. Only functional assembly can be combined with NR1 to form functional channels.
Second, it is known that NR1-1a can overcome its endoplasmic reticulum retention by assembly with NR2 and be transported to the surface of the cell membrane, but its mechanism is not clear. Our previous study tried to find a functional area to overcome the retention of the NR1a endoplasmic reticulum on NR2, and did various missing mutants, but failed to find special functional areas. In addition, we constructed various C terminal truncated plasmids of NR2B. It was found that other missing bodies could be co expressed with NR1-1a on the surface of the cell membrane except that 2BA2 could not obtain the expression of cell membrane surface with NR1-1a, and the whole cell patch clamp technique was used to record the body current of NMDA in the transfected cells. The results were in agreement with the results of surface staining. The results show that the C terminal amino acid length of NR2B has an important effect on overcoming the endoplasmic reticulum retention of NR1-1a. In other words, the retention of three amino acid lengths at the C terminal of NR2 subunit is necessary for overcoming NR1-1a's endoplasmic reticulum retention basis. In order to verify this result, we have obtained a NR1-1a-AAA mutant, only RRR hysteresis of NR1-1a. The retention of the sequence turned into AAA. and then co transfected 2B delta 2 with NR1-1a-AAA mutants to HEK293 cells, and the surface staining results showed that they were able to obtain cell membrane expression. One problem we wanted to know was whether the amino acid sequence was specific? To answer this question, we constructed the NR2B Delta 5_ (AAAAA) mutation. After CO transfection of it and NR1-1a into HEK293 cells, surface staining and electrophysiological results showed that their formed complexes could still reach the surface of the cell membrane and form a functional receptor. The results showed that the length of the three amino acids after the fourth transmembrane region of NR2B was not sequence specific. Based on the above results, the first time of this study was made. The retention of three amino acid lengths at the C terminal of the present NR2 subunit is necessary for overcoming the endoplasmic reticulum retention motif of NR1-1a. This regulation mechanism is likely to conceal the retention of the endoplasmic reticulum at the NR1-1a C terminal by initiating a change in the self allosteric structure of the C terminal of the NR1 subunit, which makes NR2 Delta 3/NR1-1a obtain the surface expression of the cell membrane. A new mechanism of NMDA receptor subunit assembly and transport is revealed.
In addition, this study uses FRET technology to observe whether the C terminal of NR2B subunit affects the assembly of NMDA receptors. The constructed YFP-2B delta 2, CFP-2B delta 2 plasmids are co transfected to HEK293 cells, and they are still able to occur FRET, which indicates that YFP-2B delta 2 and CFP-2B delta 2 can form complex. -1a was co transfected to HEK 293 cells, and it was found that they could also produce FRET.. These results suggest that the NR2B subunit missing the C terminal can still be assembled with NR1 to form a NMDA receptor complex, suggesting that the C terminal of NR2B is not a determining site for the assembly of NMDA receptors.
Finally, GFP-NR2A, GFP-NR2B, 2A Delta, 3,2A Delta 2,2B delta 3 and 2B delta 2 were transfected separately in the cultured hippocampal neurons. The expression of them in the cell membrane of the neurons was observed by living cell surface staining. The results showed that 2A Delta 5,2A Delta 3,2A delta 2 alone could reach the surface of the cell membrane, although it was significant compared with the wild type. Interestingly, it is interesting that 2B delta 2 can not be expressed in neurons alone, but NR1-4a and 2B delta 2 co transfected into neurons can observe the arrival of 2B delta 2 on the surface of the cell membrane, although there is a significant decrease in the number of membrane surface expressions compared with the wild type. It is well known that there are various endogenous NR in the neurons. 1 splice variants, if NR2 can be assembled with these different NR1 variants, then 2B delta 2 should be able to assemble and reach the surface of the cell membrane with NR1-4 subunits, but the result is that 2B delta 2 cannot reach the surface of the cell membrane. The result suggests a new phenomenon that different NR2 subunits are not randomly assembled to form NMD with various NR1 variants. The A receptor is probably different from the NR1 assembled by NR2A and NR2B.
To sum up, the main conclusions of this study are as follows: 1) it is found that the chimeras of NR2B_ (NR4aC) can be expressed on the surface of the cell membrane. Electrophysiological results suggest that NR2 subunits can not form functional ion channels alone. 2) this study first found that the length of three amino acids in the C terminal of NR2 subunit is necessary to overcome the retention of the endoplasmic reticulum of NR1-1a. It reveals a new mechanism of NMDA receptor subunit assembly and transport regulation.

【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2008
【分類號(hào)】：R33

【引證文獻(xiàn)】

相關(guān)期刊論文 前2條

1 何焰鵬;劉國(guó)榮;王寶軍;梁芙茹;鄒春燕;;N-甲基D-天冬氨酸受體NR2B亞基與學(xué)習(xí)記憶關(guān)系研究進(jìn)展[J];國(guó)際神經(jīng)病學(xué)神經(jīng)外科學(xué)雜志;2013年04期

2 盧璽宇;屈強(qiáng);;N-甲基-D-天冬氨酸受體介導(dǎo)神經(jīng)突觸長(zhǎng)時(shí)程增強(qiáng)的研究進(jìn)展[J];醫(yī)學(xué)綜述;2011年16期

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本文編號(hào)：1890632