利用細胞模式探討 GJB4 基因突變造成非症候群聽障的機制

間隙連接 ( Gap junction, GJ ) 是細胞與細胞之間的運輸通道，可以讓分子量小於 1 KDa 的分子通過。每一個 GJ 由兩個半離子通道 ( hemichannels ) 組成，而一個半離子通道則是由 6 個 connexin ( CX ) 組成。當 CX 基因，包含 GJB2、GJB3、GJB4、GJB6、GJC3 及 GJA1 發生突變時可能會造成聽力障礙。先前實驗室已經在國內 250 位非症候群聽障學童中發現到 5 個突變型的 GJB4 基因 ( CX30.3 )，分別為64C→T/wt(R22C)、109G→A/wt(V37M)、220G→A/wt(V74M)、302G→A/wt(R101H)、507C→G/507C→G(C169W)。但這些突變型 GJB4 基因的功能性尚不清楚。為了探討 GJ 的功能性是否因為突變型 GJB4 基因的影響導致 GJ 發生異常，所以我們在本研究中以不含內生性 connexin 的 HeLa 細胞作為細胞模式來觀察野生型 CX30.3 ( CX30.3WT ) 與突變型 CX30.3 ( mutant CX30.3 ) 在細胞內的表現型態。於是我們先將 CX30.3WT 與突變型 CX30.3 與綠螢光蛋白 ( LEGFP ) 融合之後，再把已融合蛋白轉染(transfection ) 到細胞中表現並使用螢光顯微鏡觀察 ( CX30.3WT ) 與突變型 CX30.3 在細胞內的表現型態。從先前實驗室的結果已知 CX30.3WT - TagRFP 融合螢光蛋白會表現在細胞質內，而在本論文的結果也可發現 CX30.3WT - LEGFP 融合螢光蛋白同樣會表現在細胞質內。且五種突變型的 CX30.3 - LEGFP 融合螢光蛋白與 CX30.3WT – LEGFP 同樣也會表現在細胞質內。而根據先前的文獻結果顯示野生型 CX31 ( CX31WT ) 會與 CX30.3WT 共同以 GJ 斑塊 ( plaque ) 的型態表現在細胞膜上。為了探討 CX31WT 是否會與先前文獻結果一致與 CX30.3WT 共同以 GJ plaque 的型態表現在細胞膜上，所以我們也同時將 CX31WT 與 CX30.3WT 共同轉染至 HeLa 細胞中觀察在細胞內的共同表現型態。從我們觀察的結果中得知野生型 CX31WT 會與 CX30.3WT 共同以 GJ plaque 的型態表現在細胞膜上，這個結果與先前文獻的結果是一致。接著我們繼續探討野生型 CX31WT 與突變型 CX30.3 在細胞內的共同表現型態。研究結果顯示突變型 CX30.3 無法與野生型 CX31WT 共同在細胞膜上表現。根據這些實驗結果，我們推測野生型 CX30.3WT 蛋白無法獨自形成 GJ ，而需與野生型 CX31WT 共同表現才會以 GJ plaque 的型態表現在細胞膜上。但突變型 CX30.3 蛋白則無法與野生型 CX31WT 蛋白共同以 GJ plaque 的型態在細胞膜上表現。關於突變型 CX30.3 的研究應該可以幫助我們了解在聽障中 GJB4 基因扮演的角色。

關鍵字

非症候群聽障；間隙連接；半離子通道；細胞模式

並列摘要

Gap junctions ( GJ ) are groups of intercellular channels that allow transport of molecules with size less than 1KD. Each GJ is composed of two hemichannels, which are themselves each constructed out of six connexin ( CX ) molecules. Mutations in the CX gene family, including GJB2, GJB3, GJB4, GJB6, GJC3, and GJA1, have been shown to underlie distinct genetic forms of hearing loss. Recently, we have identified five missense mutations in GJB4 ( CX30.3 ) ( 64C→T/wt(R22C) 、 109G→A/wt(V37M) 、 220G→A/wt(V74M) 、 302G→A/wt(R101H) 、 507C→G/507C→G(C169W) ) gene, whose defect cause hearing loss. However, the functional change in these mutants remains unknown. In this study, we want to know whether the function of GJ will be interfered by these variations. To determine the functional phenotypes of these mutations, we transfected GJ-deficient HeLa cells with WT or mutants CX30.3 fused with LEGFP. Wild-type or mutant CX30.3 protein expression in HeLa cells was analyzed by a direct fluorescent protein fusion method involving fusion of EGFP to the C-terminal ends. Mounted slides were visualized and photographed using a fluorescence microscope. According to previous studies, we have found that CX30.3WT - TagRFP fusion proteins were accumulates in the cytoplasm near the nucleus. The same results were also observed in the HeLa cells with CX30.3WT - LEGFP. Mutant CX30.3 were also accumulates in the cytoplasm near the nucleus, which is similar to the CX30.3WT. In addition, we found that cells co-expressing both CX30.3WT and the CX31WT protein exhibited co-assembly expression in the cell membrane of HeLa cells. However, we found that cells expressing both mutant CX30.3 and the CX31WT protein did not co-expressed in the cell membrane of HeLa cells. Based on these findings, we suggest that CX30.3WT proteins may form GJ between adjacent HeLa cells with CX31WT, but mutant CX30.3 did not form GJ with CX31WT. The study of CX30.3 variants should help in defining the role of CX30.3 in the hearing loss.

並列關鍵字

Nonsyndromic Deafness ； Gap Junction ； GJB4 gene ； Connexin ； CX30.3 ； hemichannel ； Cell Model

參考文獻

Bruzzone, R., White, T.W., Paul, D.L. ( 1996 ). Connections with connexins : the molecular basis of direct intercellular signaling. Eur J Biochem 238:1-27

Chika Nambara, Yumi Kawasaki, Hiroshi Yamasaki ( 2007 ). Role of the Cytoplasmic Loop Domain of Cx43 in Its Intracellular Localization and Function:Possible Interaction with Cadherin. J Membrane Biol 217:63-69

DeStefano, A.L., Cupples, L.A., Arnos, K.S., Asher, J.H., Jr., Baldwin, C.T., Blanton, S., Carey, M.L., da Silva, E.O., Friedman, T.B., Greenberg, J., Lalwani, A.K., Milunsky, A., Nance, W.E., Pandya, A., Ramesar, R.S., Read,

( 1998 ) Correlation between Waardenburg syndrome phenotype and genotype in a population of individuals with identified PAX3 mutations. Hum Genet 102:499-506

Falk, M.M., Kumar, N.M. and Gilula, N.B. ( 1994 ) Membrane insertion of gap junction connexins: polytopic channel forming membrane proteins. J Cell Biol 127:343-55

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利用細胞模式探討 GJB4 基因突變造成非症候群聽障的機制

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