| 摘要: | 甘胺酸受體 (Glycine receptor，GlyR) 為五聚體的嵌膜蛋白其所介導的抑制性神經突觸傳遞在脊椎動物的中樞神經系統扮演相當重要的角色。當 GlyR 被活化時，使得氯離子流入神經細胞中造成膜電位超極化 (hyperpolarization)，進而達到抑制神經細胞興奮以傳遞神經訊息。不同組成 GlyR 次單元 (a 或是 b) 可以在不同細胞亞結構中出現，如突觸後 (ab，異源結構)，突觸周邊或突觸前 (a，同源結構)。當 GlyR a1 發生突變時所造成部分功能缺失時，會引發一些神經相關之疾病，例如：人類的過度驚嚇症 (Hyperekplexia, Startle disease)。
在 Hyperekplexia 病人中確定 GlyR a1 突變株發病原因主要大致可區分為：GlyR a1 本身對於激動劑的親和性 (agonists affinity) 降低或是 GlyR a1 被活化後所產生的最大電流下降。在我們的研究裡，發現在 Hyperekplexia 中 GlyR a1 突變株 A384P 以及 W170S 並無上述明顯之缺失。再進一步研究中發現 A384P 突變株在不同的 agonists 活化後均有明顯之脫敏反應 (desensitization)。表現於初代培養分離性神經元細胞中 (primary dissociated neuron culture cells；cortical neurons，dorsal root ganglia neurons) 的 GlyR a1A384P 或是 a1A384P?? 同樣具有明顯的脫敏反應。另外模擬突觸遞質釋放實驗中發現 a1A384Pb 介導之突觸反應相較於 a1WTb 顯示出對低頻刺激 (1 Hz) 都具有明顯的脫敏反應後恢復延遲，這種現象隨之刺激的頻率增高而增強。因此我們推論脫敏反應引發的恢復延遲導致突觸反應減少與人類過度驚嚇症具有直接關聯性。另一方面，我們先前研究中發現 GlyR a1W170S 會特異性去除 Zn2+ 增強作用 (potentiation)；有趣的是W 和 S 都不認為會直接與Zn2+ 結合作用。目前認為 GlyR 透過 b9 片層上 E192 和 D194 以及 b10 片層上 H215 與 Zn2+ 相互作用後以達到 Zn2+ 所介導的變構調節 (allosteric modulation) 增強作用，而位於 b8 片層上的 W170 如何在此變構調節發揮作用並不清楚。因此我們利用點突變的方法對 W170 以及周圍胺基酸殘基進行改變來觀察 W170 如何在此調控過程中扮演重要的角色。我們的結果發現在 b8 片層上 170- 位點的疏水性和 Zn2+ 的調控作用呈現正相關而對於正常激動劑的敏感性有負相關的影響。只有苯環與疏水性的結構同時存在下才會完整維持 GlyR 激動劑敏感性以及 Zn2+ 正向變構調節。更進一步我們發現 b8 與 b10 以及 b8 與 b9 片層之間的連結性都會對維持激動劑的敏感性有意義而 8 和 b9 片層之間的相互作用對於 Zn2+ 正向調節更為重要。利用雙突變實驗，我們同時在 b8 的 170 位點以及 b9 的 195位點進行 cystein 的改變，DTT 會可逆性 (reversible) 的影響 Zn2+ 對 GlyR 的正向調控而不影響激動劑的敏感性。上述實驗對於 GlyR 在脫敏反應以及 Zn2+ 的變構調節機制以及和 hyperekplexia 的致病性 (causative) 連結有更進一步的了解為之後 GlyR 的相關藥物開發提供依據。
Glycine receptor (GlyR) is pentameric membrane-embed protein that mediated inhibitory synaptic transmission and play an important role in the vertebrates central nervous system. When GlyR is activated, chloride ions flow into nerve cells causing hyperpolarization of membrane potential, thereby inhibiting neuronal excitation which maintains normal neuron functions. Different components of GlyR subunit (a or b) can occur in different cell substructures, such as postsynaptic (ab form, heterologous), synaptic periphery or presynaptic (a, homologous). When GlyR is mutated and leads to the loss of some functions, it would cause neurologically related diseases, such as Hyperekplexia.
The causative reasons of GlyR a1 mutant for hyperekplexia patients can be roughly distinguished as follows: GlyR has a reduced agonist affinity or the maximum current of activated GlyR decreases. In our study, GlyR a1 mutants A384P and W170S in hyperekplexia did not have the above-mentioned obvious deficiency. With further study, mutant A384P had significant desensitization after different agonist treatments. Gly ??1A384P or GlyR ??1A384P?? expressed in primary dissociated culture neurons (cortical neurons and, dorsal root ganglia neurons) also had desensitization. In addition, compared to ??1WT???z the IPSC-like simulation of GlyR ??1A384P??-mediated synaptic responses showed a significant desensitization and recovery delay after low-frequency stimulation (1 Hz). As the frequency of stimulation was increased, the recovery delay was intensified. Therefore, we demonstrated that the recovery delay caused by desensitization would cause the decrease of synaptic response and is directly related to hyperekplexia. On the other hand, our pervious study found that GlyR a1W170S specifically abolished Zn2+ potentiation; what is more interestingly, neither W or S is thought to directly bind Zn2+. It is known that through the interaction with E192 and D194 on the b9 sheet, and H215 on b10 sheet, GlyR is able to accomplish Zn2+-mediated allosteric modulation, while it remains unclear how W170 located on b8 sheet influences Zn2+ modulation. Therefore, we used point mutation to change W170 and surrounding amino acid residues to observe how W170 plays an important role in this regulation process. Our result showed that the hydrophobicity at the 170-site on the b8 sheet was positively correlated with the Zn2+ potentiation and negatively correlated with the agonists sensitivity. Only the presence of a benzene ring and a hydrophobic structure at the same time, would completely maintain GlyR agonist sensitivity and positive allosteric regulation of Zn2+. Further, we found that the connection between b8 and b9 or b8 and b10 sheet is meaningful for maintaining agonist sensitivity while the interaction between b8 with b9 sheet is more important for Zn2+ positive regulation. Using the double mutation experiment, we simultaneously performed cystein changes at the 170 site of b8 and the 195 site of bb9. DTT reversibly affected the positive regulation of Zn2+ on GlyR without affecting agonist sensitivity. These experiments provided further understanding of the desensitization responses of GlyR, the allosteric regulatory mechanisms of Zn2+ and the causative linkages with hyperekplexia, which becomes a ground for subsequent drug development in GlyR. |
