| 摘要: | Connexins (CXs) 基因族在許多研究已經被報導和非症候群遺傳性聽障有關。在我們實驗室先前針對台灣非症候群聽障病人中的篩檢，已經發現在GJC3基因 (CX30.2/CX31.3) 中有三個錯意突變點包括p.R15G, p.L23H及p.W77S。另外，我們實驗室先前用免疫組織染色法發現，鼠的耳蝸表現Cx29 (同義於人的CX30.2/CX31.3) 與其他Cxs蛋白家族成員 (如Cx26及Cx30) 所表現的位置相似，且這些位置對於維持耳蝸內的鉀子再循環相當重要。然而到目前為止對於我們所發現的這些錯意突變，所造成的影響及致病機轉並清楚，且對於CX30.2/CX31.3蛋白的主要功能亦清楚。因此在本研究中我們將加以探討。本文將分部分探討：第一部分我們用tet-on雙向表現系統，探討這些突變蛋白在tet-on HeLa細胞內的表現位置，並分析是否造成CX30.2/CX31.3蛋白的功能喪失。在我們的研究當中，我們發現含p.R15G或p.L23H的CX30.2/CX31.3突變蛋白，其表現位置和正常的CX30.2/CX31.3蛋白一樣可被運送到細胞膜上。然而含p.W77S的CX30.2/CX31.3突變蛋白則展現出運送功能常，無法正常送至細胞膜上表現，而堆積在內質網上。在共同表現蛋白的研究中，含p.W77S的突變和正常CX30.2/CX31.3蛋白共同表現時，我們發現突變蛋白會影響正常蛋白運送到細胞膜上，而一起堆積在細胞核附近。因此，我們認為含p.W77S的CX30.2/CX31.3突變蛋白對於正常CX30.2/CX31.3蛋白展現出顯性抑制效應 (dominant negative effect)，而導致正常CX30.2/CX31.3蛋白堆積在細胞質，損害hemichannels及GJ通道的形成。而含p.R15G或p.L23H的CX30.2/CX31.3突變蛋白並沒有影響其運送至細胞膜的能，然而在細胞膜上的功能是否正常仍需進一步的探討。第二部分我們想解正常CX30.2/CX31.3蛋白的運送徑及在細胞膜上形成hemichannel和GJ通道的特性。藉由免疫螢光染色法，發現CX30.2/CX31.3融合螢光蛋白會續的表現在細胞膜上，而和一般的CX蛋白家族成員會在細胞間形成斑塊的現象同。我們用藥物處的方式，將表現CX30.2/CX31.3蛋白的tet-on HeLa細胞加入Brefeldin A破壞高基氏體，發現並影響CX30.2/CX31.3的正常表現特徵，此結果與其他CXs並相同。然而，加入nocodazole破壞微管後，可明顯的影響CX30.2/CX31.3 運送到細胞膜的能。當加入cytochalasin B破壞肌動蛋白絲後，發現會些微的影響CX30.2/CX31.3 在細胞膜上的穩定表現。由以上結果我們認為，CX30.2/CX31.3蛋白在細胞內的運送過程中，並需要依賴高基氏體的修飾，主要是藉由微管運送至細胞膜上，再由肌動蛋白絲扮演穩固CX30.2/CX31.3蛋白在細胞膜上的角色。另外，進一步分析GJ通道特性時，我們發現染劑並無法在穩定表現CX30.2/CX31.3蛋白的HeLa細胞間通透。然而其hemichannels在低鈣濃下，可以有意義的釋放ATP至細胞外。因此，我們認為CX30.2/CX31.3蛋白與Pannexin蛋白功能相似，主要扮演的功能角色為hemichannel功能，而是像一般CX蛋白所形成的GJ通道功能。
Connexins (CXs) are known to be involved in human nonsyndromic genetic deafness. Among a cohort of patients with nonsyndromic hearing loss, we recently identified novel heterozygous missense mutation, p.R15G, p.L23H and p.W77S, in the GJC3 gene encoding CX30.2/CX31.3, as being causally related to hearing loss. In addition, by using IHC analysis in our previous study, mouse Cx29, orthologs of human CX30.2/CX31.3, just like other Cxs (Cx26 and Cx30), were found in many parts of the cochlea along the proposed K+ recycling pathway. The functional alteration of CX30.2/CX31.3 caused by the mutant GJC3 gene, however, remained unknown. In addition, the functional characteristics of CX30.2/CX31.3 are unclear. We divided this thesis into two part：in the first part, we compared the mutant intracellular distribution and effect on tet-on HeLa cells. Our protein localization result indicted that p.R15G and p.L23H mutant exhibited continuous fluorescence along the apposed cell membranes by immunofluorescent assay, which is similar to the wild-type (WT). However, p.W77S mutant showed impaired trafficking of the protein to the plasma membrane and accumulation in the endoplasmic reticulum (ER). In co-expression study, p.W77S mutant co-expressed with WT which displayed both of them were perinuclear localization, which impairment of the ability of both proteins to targeting to the plasma membrane. In summary, p.W77S mutant exhibit the dominant negative effect on normal CX30.2/CX31.3 leading to accumulation of the CX proteins in the cytoplasm that impairs formation of hemichannels and GJ. But the p.R15G and p.L23H mutant do not affect the trafficking of CX proteins. The functional significance of p.R15G and p.L23H requires further investigation. In the second part, we assessed normal CX30.2/CX31.3 trafficking and function properties on hemichannels and GJ. In the immunofluorescent assay, unlike other CXs, CX30.2/CX31.3-EGFP exhibited continuous fluorescence along the apposed cell membranes instead of punctated fluorescence in contacting membranes. Brefeldin A, a Golgi apparatus disruptor, does not affect CX30.2/CX31.3 protein in the ability of intracellular trafficking and targeting to plasma membrane, but has effect on other CX protein. However, both the pattern and the distribution of CX30.2/CX31.3 were predominantly affected by nocodazole, which can dissemble the microtubules. In addition, the targeting to channels of CX30.2/CX31.3 was minimally affected by cytochalasin B, an inhibitor of actin polymerization. Based on these findings, we suggest that CX30.2/CX31.3 travel to the plasma membrane to form channels by Golgi-independent secretory pathway. The cytoskeletons, especially microtubules, are important components in the processes of assembly and trafficking of CX30.2/CX31.3, and the docking in the membrane as likely depends on the actin filaments. To investigate the function properties on GJ, surprisingly, dyes were impermeated by CX30.2/CX31.3 GJ, but there was a significant release of ATP from HeLa cell line stably expressing CX30.2/CX31.3 hemichannel, in medium with low calcium ion concentration. Therefore, we suggest that CX30.2/CX31.3 shares functional properties with pannexin (hemi) channels rather than gap junction channels of other CXs. |
