發(fā)布時間：2018-08-27 12:51

【摘要】：NMDA受體是由NR1和NR2亞單位組成的異聚體復合物,亞單位之間的裝配對于NMDA受體通道的形成和表面表達至關(guān)重要。以往的研究提示,NR2亞單位胞內(nèi)C末端區(qū)可能參與調(diào)節(jié)NMDA受體的表面表達和突觸定位。本研究的第一部分通過一系列C末端截短的NR2A亞單位突變體的構(gòu)建以及NMDA受體膜表面表達和功能的檢測,在HEK293細胞和培養(yǎng)海馬神經(jīng)元研究了NR2A亞單位的C末端在NMDA受體裝配和表面表達中的作用。結(jié)果發(fā)現(xiàn),當NR2A亞單位的C末端截短后其TM4下游分別剩下59、5、3個氨基酸殘基時(2AΔC59、2AΔC5和2AΔC3),它們均能與NR1-1a形成有功能的受體通道并表達于細胞膜表面,但是與未截短的NR2A相比其表面表達均有所下降;并且,當2AΔC5的TM4下游僅存的5個氨基酸EHLFY突變成EAAAA(2AΔC5_(EAAAA))時,仍可與NR1-1a形成受體復合物并表達于細胞膜表面。若將NR2A的C末端截短僅保留TM4后的1個氨基酸時(2AΔC1),該突變體與NR1-1a共轉(zhuǎn)染時,就不再獲得受體復合物的細胞膜表面表達,也不能記錄到谷氨酸誘發(fā)的電流。有趣的是,當NR1-4a與2AΔC1共轉(zhuǎn)染時,NR1-4a/2AΔ1受體復合物仍然能表達于HEK293細胞表面,并能檢測到通道功能。在此,與NR1-1a不同的是NR1-4a不含有內(nèi)質(zhì)網(wǎng)滯留基序。以上結(jié)果提示,NR2A亞單位TM4后的3個氨基酸是NR1-1a/NR2A受體復合物細胞膜表面表達所必需的,然而,NR2A亞單位的C末端并不是NR2A與NR1裝配和形成功能性通道所必需的,但能幫助NR1-1a克服內(nèi)質(zhì)網(wǎng)滯留作用使NMDA受體復合物表達于細胞膜表面。 本研究的第二部分研究了NR2A亞單位單獨轉(zhuǎn)染時膜表面表達。以往研究
[Abstract]:NMDA receptor is an heteropolymer complex composed of NR1 and NR2 subunits. The assembly between subunits is very important for the formation and surface expression of NMDA receptor channels. Previous studies suggest that the C-terminal region of NR2 subunit may be involved in regulating the surface expression and synaptic localization of NMDA receptor. In the first part of this study, a series of C-terminal truncated NR2A subunit mutants were constructed and the surface expression and function of NMDA receptor were detected. The role of C-terminal of NR2A subunit in the assembly and surface expression of NMDA receptor was studied in HEK293 cells and cultured hippocampal neurons. The results showed that when the C-terminal of NR2A subunit was truncated, the downstream of TM4 remained 59,5 and 3 amino acid residues (2A 螖 C59C5 and 2A 螖 C3), which could form a functional receptor channel with NR1-1a and express on the surface of cell membrane. However, compared with the untruncated NR2A, the expression of EHLFY on the surface of TM4 of 2A 螖 C5 was decreased, and when the only 5 amino acid EHLFY of TM4 of 2A 螖 C5 mutated to EAAAA (2A 螖 C5 _ (EAAAA), it could form receptor complex with NR1-1a and express on the surface of cell membrane. If the C-terminal of NR2A was truncated with only one amino acid (2A 螖 C1) of TM4, the expression of the receptor complex on the cell membrane and the glutamate induced current could not be recorded when the mutant was co-transfected with NR1-1a. Interestingly, when NR1-4a was co-transfected with 2A 螖 C1, the NR1-4a / 2A 螖 1 receptor complex could still be expressed on the surface of HEK293 cells and the channel function could be detected. Here, unlike NR1-1a, NR1-4a does not contain endoplasmic reticulum retention motifs. These results suggest that the three amino acids of NR2A subunit after TM4 are necessary for the surface expression of NR1-1a/NR2A receptor complex. However, the C-terminal of NR2A subunit is not necessary for the assembly and formation of functional channels between NR2A and NR1. But it can help NR1-1a overcome endoplasmic reticulum retention and make NMDA receptor complex express on the surface of cell membrane. In the second part of this study, the membrane surface expression was studied when NR2A subunits were transfected alone. Previous research
【學位授予單位】：浙江大學
【學位級別】：博士
【學位授予年份】：2005
【分類號】：R33

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本文編號：2207339