分析吳郭魚 AMPA 受器次單元 tfGRIA1 去敏感化作用

Analysis of the desensitization of a tilapia AMPA receptor subunit tfGRIA1

林煒翔(Wei-Hsiang Lin)

指導教授 ： 周姽嫄

國立清華大學/生命科學院/生命科學系/博士(2006年)

https://doi.org/10.6843/NTHU.2006.00384

摘要

AMPA 受器廣泛表現於哺乳動物的中樞神經系統，是中樞神經中主要的興奮性傳導物質接受器。透過 AMPA 受器 C 端序列與結合蛋白的交互作用，調控受器在神經突觸上的表現、聚集以及在細胞膜與細胞質之間的輸送。硬骨魚類由於基因複製的關係，斑馬魚及吳郭魚皆表現八種 AMPA 受器次單元。演化過程中，魚類 AMPA 受器的胺基酸序列及功能雖皆具保留性，然而仍存有些許變異。我們以酵母菌雙合實驗篩選出吳郭魚 AMPA 受器次單元 tfGRIA1β C 端序列交互作用蛋白 p22。p22 帶有 4 個 EF-hand 鈣離子結合位及 N-myristoylation 修飾位。破壞 p22 第 3 個 EF-hand 鈣離子結合位喪失與 tfGRIA1β C 端序列結合的能力，而破壞 p22 N-myristoylation 修飾位不影響與tfGRIA1β C 端序列的結合。tfGRIA1β 與 p22 結合是透過tfGRIA1β C 端 28 個胺基酸。比較 tfGRIA1β 與其他能與 p22 結合的蛋白質序列，顯示 p22 不是透過特定序列與 tfGRIA1β C 端結合。我們的實驗也證明 tfGRIA1β C 端與 p22 結合是透過一段能形成 amphipathic α-helix 結構的胺基酸序列以及周圍相鄰序列。過量表現 p22 促使帶有 tfGRIA1β C 端序列的受器嵌合體 tfGRIA3αi/1β N21 離子通道 Glu/KA 斜率趨勢改變，且趨勢的改變受到減緩去敏感化作用的藥物 cyclothiazide 所抑制，顯示 p22 參與 AMPA 受器調控去敏感化作用機制。這是目前所知透過蛋白質交互作用影響 AMPA 受器去敏感化作用的第一個例子。
AMPA 受器次單元中之 flip/flop 區域也參與調控受器去敏感化作用機制。tfGRIA1α 次單元 flip 序列與多數脊椎動物 AMPA 受器次單元 flip 序列差異較大。我們也證明 tfGRIA1αflip 中 I740 胺基酸參與調控受器的去敏感化速率，然而單一改變 tfGRIA3αflip T740 成為 I740 不影響離子通道去敏感化速率，顯示 AMPA 受器去敏感化速率非單藉由 I740 胺基酸決定。

關鍵字

麩胺酸受器 ； 去敏感化作用

並列摘要

AMPA receptors (AMPARs) are major excitatory neurotransmitter receptors and are wildly expressed in the mammalian central nervous system. Through interactions with cellular proteins, the C-terminal domains of AMPAR subunits regulate membrane expression, clustering and recycling of the receptors. Due to a teleost-specific gene duplication event, both zebrafish and tilapia express 8 AMPAR subunits. The primary sequences and ion channel functions are conserved between mammalian and teleost AMPAR subunits. However, some interacting protein recognition sequences of teleost AMPAR subunits are different from that of mammalian AMPAR subunits. An AMPAR subunit tilapia tfGRIA1β C-terminal interacting protein p22 was identified by yeast two-hybrid system. The structure of p22 contains 4 EF-hand calcium binding domains and an N-myristoylation site. Disruption of the third EF-hand domain of p22 affected the interactions with tfGRIA1β C-terminal sequence, whereas disruption of the N-myristoylation site did not affect the interaction. Interaction between tfGRIA1β C-terminal domain and p22 was mapped to a stretch of 28 amino acid sequence which displayed very limited similarities to the interaction sequences found in the other known p22 binding proteins. The lack of sequence conservation among p22 interacting proteins suggests that ligand recognition by p22 is not mediated through consensus sequence. Our result indicated that amphipathic α-helix structure and its neighboring sequences are required for the interaction between p22 and tfGRIA1β subunit. Overexpressing p22 resulted in the alteration of the ratios between maximum currents induced by glutamate and by kainate (IGlu/IKA) of a chimera tfGRIA3αi/1β N21 receptor. The glutamate-induced current is subjected to desensitization, whereas kainate-induced current is not. Addition of the desensitization attenuator, cyclothiazide, abolished the p22 effects on alternation of IGlu/IKA, suggesting that p22 is involved in regulating the desensitization of tfGRIA3αi/1β N21 receptor. This is the first report of regulating AMPAR desensitization accomplished by direct protein-protein interaction.
The flip/flop region of AMPAR subunits involves in the regulation of desensitization. The flip sequence of teleost GRIA1α subunit varies from that of the other vertebrate AMPAR subunit. We demonstrated that the I740 residue of tfGRIA1αflip subunit involved in the regulation of receptor desensitization. However, mutating the T740 residue to I740 of tfGRIA3αflip subunit does not affect the desensitization, indicating that desensitization is not determined by the Ile at the position 740 alone.

並列關鍵字

Glutamate receptor ； desensitization

參考文獻

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