非症候群聽障中突變的Cx26和Cx30對正常的Cx26和Cx30所造成的影響

人類的gap junction 蛋白 connexin26 (Cx26) 和connexin30 (Cx30)產生突變會導致聽障。 Cx26 和Cx30會在耳蝸中共同形成 heteromeric 的半通道，以及heterotypic 的gap junction 通道。我們實驗室先前在台灣非症群聽障病人中篩選出GJB2 368C>A (Cx26 T123N), GJB2 551G>A (Cx26 R184Q) 和GJB6 119C>T (Cx30A40V) 這三個突變點，他們都是heterozygous。為了去研究這三個heterozygous 突變點所造成的影響，我們利用HeLa 細胞株使和tet-on 蛋白表現系統結合螢光蛋白系統，單獨來表現正常或是突變的Cx26 和Cx30，以及把正常Cx26 或Cx30 和三個突變點一起共同表現來觀察他們彼此間的交互影響。在我們的研究當中，突變點Cx26 T123N 很明顯表現在細胞膜上，這和正常Cx26 或Cx30所表現的結果是一樣的。突變點Cx30 A40V 和Cx26 R184Q 則展現出運送功能異常，無法將其蛋白送達細胞膜上，而堆積在高基氏體和內質網上。而在共同表現蛋白的研究當中，Cx26 R184Q 或Cx30 A40V 和正常的Cx26 或Cx30共同表現時，結果顯示正常和突變的Cx 蛋白都堆積在細胞核附近，我們推測此兩種突變蛋白並無法運送至細胞膜。因此會影響正常的Cx26 和Cx30蛋白在細胞內蛋白運輸以及送往目標細胞膜的功能。相反的，當Cx26 T123N 和正常Cx26 或Cx30蛋白共同表現時，顯現出和正常Cx26 或Cx30蛋白相似的結果。綜合上面的結果，Cx26 R184Q ，Cx30 A40V 對於正常Cx26 和Cx30正常Cx30的蛋白展現出 cis- 或trans- dominant negative 的影響，而導致正常Cx26 或Cx30 的蛋白堆積在細胞質，損害gap junction 的形成。而突變基因Cx26 T123N 則對於Cx 蛋白沒有產生影響。Cx26 T123功能上的重要性仍需進一步的實驗去證實。

Mutations in the human gap junction proteins connexin26 (Cx26) and connexin30 (Cx30) cause hearing loss. Cx26 and Cx30, expressed in the cochlea, form heteromeric hemichannels and heterotypic gap junction channels. Three heterozygous mutations, GJB2 368C>A (Cx26 T123N), GJB2 551G>A (Cx26 R184Q) and GJB6 119C>T (Cx30A40V), were identified in our previous study from Taiwanese non-syndromic deafness patients. To investigate the functional consequence of these heterozygous mutations, we expressed individual wild-type or mutant Cx26 and Cx30 and co-expressed human Cx26wt/mutant or Cx30wt/mutant in HeLa cells by tet-on expression system with fluoresce fusion protein system. In our study, whereas Cx26 T123N showed predominantly membrane localization, which is similar to the characteristic punctuate expression in plasma membrane of the wild-type Cx26 and Cx30, Cx30 A40V and Cx26 R184Q showed impaired trafficking of the protein to the plasma membrane and accumulation in the Golgi or ER. In co-expression study, Cx26 R184Q or Cx30 A40V co-expressed with either Cx26 or Cx30 protein displayed both of them were perinuclear localization, suggesting affect wtCx26 or wtCx30 impairment of the ability of both proteins to intracellular trafficking and targeting to the plasma membrane. In contrast, Cx26 T123N co-expression with either Cx26 or Cx30 exhibited membrane localization similar to that of wild-type Cx26 or Cx30. In summary, Cx26 R184Q and Cx30 A40V exhibit cis- or trans-dominant negative effect on both normal Cx26 and Cx30 leading to accumulation of the Cx proteins in cytoplasm that impairs formation of the gap junction. Cx26 T123N does not affect the trafficking of Cx proteins. The functional significance of Cx26 T123N requires further investigation.