E3 泛素連接酶與 helix O 突變對人類 CLC 通道之功能效應

Abstract

人類第一型及第二型氯離子通道(ClC-1, ClC-2)具有重要的生理功能。在正常的骨骼肌細胞中， ClC-1 主要負責維持細胞膜電位的穩定，當 ClC-1 產生突變時可能會導致先天性肌強直症(myotonia congenita)的發生；而 ClC-2 則是參與了多種不同的生理調控機制，包括腸胃道、細精管以及細胞的體積大小、酸鹼值等等，也有研究指出 ClC-2 與腦白質失養症、原發性高醛固酮症、無精症等疾病有關。細胞蛋白質的恆定會受到多種不同的內質網品質控管途徑來調控，而泛素-蛋白酶體系統能夠將專一性的泛素標定到目標蛋白上，再經由蛋白酶體使目標蛋白降解。本實驗室先前發現多種 E3 泛素連接酶與 ClC-1 及 ClC-2 具有交互作用，故本文會以電生理的角度來觀察 E3 泛素連接酶是否實際參與膜上具有功能的 ClC-1 及 ClC-2 蛋白恆定之調控，進而改變氯離子電流密度(Cl- current density)大小。根據我的實驗結果顯示，若大量表現 RING finger-like E3 ligases 或 HECT domain E3 ligases 時皆會影響其電流表現，因此我們認為這些 E3 泛素連接酶可以經由影響氯離子通道的蛋白恆定，繼而調控細胞膜上 ClC-1 及 ClC-2 之功能表現。此外，先前實驗室發現 ClC-1 及 ClC-2 的 helix O 可能參與其蛋白質穩定，其他文獻也曾報導肌強直症相關的 ClC-1 突變 A531V 及 G523D 位於 helix O 上，其中A531V 會造成蛋白表現量減少， G523D 則改變了通道的電壓依賴性；而與腦白質失養症有關的 ClC-2 突變 A500V 及 G503R 亦位於 helix O 上。因此，我們分別將 ClC-1 及 ClC-2 的 helix O 部分位點進行突變，並利用電生理技術探討 helix O 對通道之影響。我們發現 ClC-1 helix O 上的突變使得量測到的電流皆小於在其他 helix 上的肌強直症突變；此外也發現 ClC-2 上的 helix O 突變會使電流減少許多。據此我們可以推知此類型氯離子通道的 helix O 極大可能影響其蛋白能否穩定表現在細胞膜上，抑或是影響其從內質網運送至細胞膜之效率。

Human ClC-1 and ClC-2 chloride channel play an important role in physiological functions. In normal skeletal muscle cells, ClC-1 is mainly responsible for maintaining the stability of the cell membrane potential. When ClC-1 mutated, it may lead to abnormal function of the channel protein and then cause myotonia congenita. ClC-2 is involved in a variety of different physiological regulatory mechanisms, including gastrointestinal tract, seminiferous tubules, cell’s size, cell’s pH value, etc. In addition, some studies have pointed out that ClC-2 is related to leukodystrophy, primary aldosteronism and azoospermia. Proteostasis is regulated by a variety of endoplasmic reticulum quality control pathways. The ubiquitin-proteasome system can target ubiquitin to the target protein specifically, and then degrade the target protein via the proteasome. Our laboratory has previously discovered that various of E3 ubiquitin ligases interact with ClC-1 and ClC-2. Therefore, in this study, we aim to apply electrophysiological techniques to observe whether E3 ubiquitin ligases are involved in functional ClC-1 and ClC-2 on the cell membrane. According to my experimental results, RING finger-like E3 ligases or HECT domain E3 ligases affected the current of chloride channels. Therefore, we believe that E3 ubiquitin ligase can regulate the functional expression of ClC-1 and ClC-2 on cell membrane by affecting the protein stability of the chloride channels. In addition, previous laboratories have found that helix O of ClC-1 and ClC-2 may be involved in their protein level. Other paper also reported that myotonia-related ClC-1 mutations A531V and G523D are located on helix O. A531V causes protein level reduced and G523D changes the voltage dependence of the channel. ClC-2 mutations A500V and G503R associated with leukodystrophy are also located on helix O. Therefore, we mutated some of the helix O sites in ClC-1 and ClC-2, and then used electrophysiological techniques to explore the effect of helix O on the channels. We found that the mutations on ClC-1 helix O caused the current smaller than that of myotonia mutations on other helixes. We also found that the helix O mutations on ClC-2 reduced the current. Based on this, we can infer that the helix O of ClC chloride channel may greatly affect whether its protein can be stably stored on the cell membrane or affect the efficiency of transport from the endoplasmic reticulum.