利用細胞模式探討GJC3突變造成非症候群聽障之功能研究

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研究生：	梁瑋光 Liang, Wei-Guang
論文名稱：	利用細胞模式探討GJC3突變造成非症候群聽障之功能研究 Investigation the GJC3 mutant caused nonsyndromic hearing loss using cell model
指導教授：	江安世 Chiang, Ann-Shyn 李宣佑 Li, Shuan-Yow
口試委員:	江安世李宣佑謝明麗蕭光明楊建洲
學位類別：	博士 Doctor
系所名稱：	生命科學暨醫學院 - 生物科技研究所 Biotechnology
論文出版年：	2011
畢業學年度：	99
語文別：	英文
論文頁數：	110
中文關鍵詞：	非症候群聽障、GJC
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中文摘要

細胞間隙接合溝通有許多功能，每一種功能皆是為了滿足組織、器官，或是一群細胞的特別需求。間隙連接通道是由連接蛋白connexin (CX) 組成的，每個connexin連接蛋白有四個穿膜區且在許多研究已經被證實和聽覺的功能有關。CX30.2/CX31.3 (GJC3)在CX家族中是屬於較新的成員。我們先前利用免疫組織染色法發現，老鼠的耳蝸表現Cx29 (同義於人類的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離子再循環相當重要。然而到目前為止我們對於CX30.2/CX31.3蛋白的主要功能尚不清楚。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) 中有兩個錯意突變點包含p.R15G及p.L23H。而我們對於所發現的這兩個錯意突變所造成的影響及致病機轉並不清楚。
因此在本研究中我們將分兩部分來探討：第一部分，我們想了解正常CX30.2/CX31.3蛋白的功能，包括在HeLa細胞的表現位置，能否有效地將染劑通透及形成間隙接合通道的特性。藉由免疫螢光染色法發現，CX30.2/CX31.3融合螢光蛋白會連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不同。進一步分析間隙接合通道特性時，我們發現大部分染劑並無法在穩定表現CX30.2/CX31.3蛋白的HeLa細胞間通透，這與先前研究發現CX26可以通透染劑的情況不同。此外，在低鈣濃度下穩定表現CX30.2/CX31.3蛋白的HeLa細胞可以增加釋放ATP至細胞外。因此，我們認為CX30.2/CX31.3蛋白主要扮演的功能角色為hemichannel功能。
第二部分，我們比較正常與含p.R15G及p.L23H突變的CX30.2/CX31.3蛋白在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。利用免疫螢光染色法發現，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蛋白一樣會連續的表現在細胞膜上，並沒有影響其運送至細胞膜的能力。進一步我們發現在低鈣濃度下，含p.R15G或p.L23H突變基因的HeLa細胞比正常表現的細胞釋放更少的ATP至細胞外。因此，我們建議p.R15G和p.L23H這兩個突變確實會影響CX30.2/CX31.3蛋白的功能，進而可能造成聽障。
綜合以上結果，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，而不是像一般CX蛋白所形成的間隙接合通道功能。我們對於CX30.2/CX31.3蛋白在聽覺所扮演的功能有進一步的了解，這或許也提供了一些診斷的價值及治療的新方向。

Abstract

Gap junctional intercellular communication (GJIC) has numerous functions, each of which meets the particular needs of organs, tissues, or groups of cells. As a component of gap junction (GJ) channel, connexin (CX) is homologous four-transmembrane-domain proteins, with numerous studies confirming its auditory functions. CX30.2/CX31.3 (GJC3) is a relatively new member of the CX protein family. 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. Until now, however, the functional characteristics of CX30.2/CX31.3 have been unclear. Among a cohort of patients having incurred nonsyndromic hearing loss, we identified two novel missense mutations, p.R15G and p.L23H, in the GJC3 gene encoding CX30.2/CX31.3, as causally related to hearing loss in previous study. However, the functional alteration of CX30.2/CX31.3 caused by the two mutants of GJC3 gene remains unknown.
We divided this thesis into two parts：In the first part, to elucidate the properties of CX30.2/CX31.3 channels, their subcellular localization in HeLa cells, their effectiveness in dye transfer, and function on channels were investigated. In the immunofluorescent assay, cells that stably expressed CX30.2/CX31.3-EGFP exhibited continuous fluorescence along the apposed cell membranes, rather than punctated fluorescence in contacting membranes between two cells. To investigate the function properties on GJ, surprisingly, dyes that can be capable of being permeated by CX26 GJ, according to a scrape loading dye transfer assay in previous studies, are impermeated by CX30.2/CX31.3 GJ, suggesting a difference between the characteristics of CX30.2/CX31.3 GJ and CX26 GJ. Furthermore, a significant amount of ATP was released from the HeLa cells that stably expressed CX30.2/CX31.3, in a medium with low calcium ion concentration, suggesting a hemichannel-based function for CX30.2/CX31.3.
In the second part, we compared the intracellular distribution of mutant CX30.2/CX31.3 (p.R15G and p.L23H) with the wild-type CX30.2/CX31.3 in HeLa cells and the effects of the mutant protein had on those cells. Analytical results indicated that p.R15G and p.L23H mutant exhibited continuous staining along apposed cell membranes in the fluorescent localization assay, which is the same with the wild type. The p.R15G and p.L23H mutant do not affect the trafficking of CX proteins. Moreover, ATP release assay results demonstrated that ATP release (hemichannel function) is less in HeLa cells carrying mutant GJC3 genes than those of wild type-expressing cells. We believe that although p.R15G and p.L23H mutants do not decrease the trafficking of CX proteins, mutations in GJC3 genes result in a loss of function of CX30.2/CX31.3 protein, possibly causing hearing loss.
In conclusion, the work in this thesis provides information for understanding the function of CX30.2/CX31.3 in nonsyndromic deafness. These data together suggest that, unlike most gap junction channels of other CXs, CX30.2/CX31.3 may act as pannexin (hemi) channels and may provide some diagnostic values and therapeutic implications in hearing loss.

Contents
Table of Contents……………………………………………...………..…………….1
Abbreviations………………………………………………………………………7
中文摘要……………………………………………………………………………..9
Abstract…………………………………………………………………………….12
Chapter 1
General introduction
1-1 Introduction to how hearing work……………………………….…………….15
1-2 Introduction of hearing loss…………………………………..…..………...…18
1-3 Gap junction in the cochlea…………………………………………...……….20
1-4 Connexin expression in inner ear and gap junction function …………….…...23
1-5 Introduction of connexin……………………………………………………... 25
1-6 Connexin mutations and deafness……………………………………………..28
1-7 Specific aims………………………………………………………...………...31
1-8 Figures
Figure 1. Schematic illustration of the human ear.……………………..…….32
Figure 2. Schematic illustrations of portions of the inner ear………………33
Figure 3.    Schematic illustration of a potassium ion recycling mechanism in the mammalian cochlea.……………………………....…..……….…....34
Figure 4.    Intercellular Ca2+-wave propagation in non-excitable cells involves both gap junctional intercellular communication (GJIC) and paracrine intercellular communication (PIC).……………………..…………..35
Figure 5. Membrane topology of connexins………………...…………………36

Figure 6.    Schematic representation of the formation of gap junction (GJ) channels and hemichannels………………………………………......37
Figure 7. Structure and properties of connexin channels………………….….38
Figure 8. The cytoskeleton is involved in different steps of connexin turnover..39
Figure 9. A diagram showing the location of reported human CX26 mutations in the CX26 protein that are linked to hearing loss……………………..40
Figure 10. Summary of the expression patterns of Cx26, Cx30, Cx31, Cx43, and Cx29 in mouse cochlea……..…………………………………….41
Chapter 2
Human connexin30.2/31.3 (GJC3) does not form functional gap junction channels but causes enhanced ATP release in HeLa cells
2-1 Introduction ……………………………………………………………….......42
2-2 Materials and methods
2-2-1 Wild-type GJC3 gene EGFP expression constructs……………...……44
2-2-2 Expression of CX30.2/CX31.3 gap junction proteins in HeLa cells…..45
2-2-3 Immunofluorescence staining of post-transfection HeLa cells……......46
2-2-4 Reverse transcription-polymerase chain reaction (RT–PCR)……….…46
2-2-5 Scrape loading dye transfer ………………………………………...…47
2-2-6 ATP release assay…………………………………………..………….48
2-3 Results
2-3-1 Construction and selection of EGFP transfected clone……………….…49
2-3-2 The CX30.2/CX31.3WT expressed in cell membrane……………..….49
2-3-3 CX30.2/CX31.3 forms functional GJ channels different from CX26…...50

2-3-4 CX30.2/CX31.3 can form calcium-dependent hemichannels and release        ATP…………………………………………………...………..……..…50
2-4 Discussion
2-4-1 Other similar CX………………………………………………………52
2-4-2 CX30.2/CX31.3 lack permeability with low molecular weight……..…..53
2-4-3 CX30.2/CX31.3 hemichannels were activated at low calcium concentrations………………………………………………...………….53
  2-5 Tables
     Table 1. ATP release concentration in the Mock HeLa and CX30.2/CX31.3 stably expression HeLa cells…………………….…………….……55
2-6 Figures
Figure 11. Expression analysis of GJC3 mRNA in the transfected HeLa cells by RT–PCR.…………………………………………………..…...56
Figure 12. Analysis of expression of CX30.2/CX31.3WT in stably transfected HeLa cells based on immunocytochemistry using pan-cadherin antibody…………………………………………………………....57
Figure 13. Dye transfer after scrape loading HeLa cells that stably express CX30.2/CX31.3.…………………………………………………..58
Figure 14. Dye transfer after scrape loading HeLa cells that stably express CX30.2/CX31.3…….……………………………………………..59
Figure 15. Analysis of activity of CX30.2/CX31.3 hemichannels based on ATP release…………………...………………………………………....60
Figure 16. The effect of 18A-GA on extracellular ATP levels…………...…...61
Chapter 3
Elucidating the contribution of two novel missense mutations in human GJC3 gene (connexin30.2/31.3) to nonsyndromic hearing loss
3-1 Introduction ………………………………...…………………………….......62
3-2 Materials and methods
3-2-1 Gene cloning and construction of the plasmids expressing wild-type or mutants CX30.2/CX31.3.…………..………..……………..……….…64
3-2-2 Cell culture and transfection…………………………....…...………....65
3-2-3 Immunofluorescence Staining of HeLa cells…………...………...……65
3-2-4 Extracellular ATP measurements……….…………...…….……..……66
3-3 Results
3-3-1 The domain structure of the CX30.2/CX31.3 protein………..……..…67
3-3-2 Alignment of the amino acid sequences of the average domain of human CX30.2/CX31.3………………………………………………….….….67
3-3-3 Construction and selection of EGFP transfected clone……………..…68
3-3-4 The mutant CX30.2/CX31.3 expressed in cell membrane………….…69
3-3-5 The mutant CX30.2/CX31.3 does not form functional channel….........69
3-4 Discussion
3-4-1 The possible reason why p.R15G mutant loss of function ……………71
3-4-2 The possible reason why p.L23H mutant loss of function…………….72
3-4-3 CX30.2/CX31.3 hemichannels were activated at low calcium concentrations………………………………………………………….73


3-5 Tables
Table 2. ATP release concentration in the WT and mutant CX30.2/CX31.3 stably expression HeLa cell…………...…………...…….….……74
Table 3. ATP release concentration in the low Ca2+ condition after 18A-GA treatment………………………………………………………… 75
3-6 Figures
Figure 17. Schematic representation of the domain structure of the CX30.2/CX31.3 protein with indication of known variants…...76
Figure 18.  Alignment of the amino acid sequences of the average domain of human CX30.2/CX31.3 and members of human CX family…..77
Figure 19. ConSeq predictions demonstrated on human CX30.2/CX31.3 (NP_853516; SWISS-PROT Q8NFK1 (CXG3_Human), using 50 homologues obtained from the Pfam database (family code: PF00029)…………..…………………………………………78
Figure 20. Expression analysis of CX30.2/CX31.3 mRNA in the three transfected HeLa cells by RT-PCR…………….....................…79
Figure 21.     Expression analysis of CX30.2/CX31.3 WT, CX30.2/CX31.3 R15G and CX30.2/CX31.3 L23H in stably transfected HeLa cells by immunocytochemistry using pan-cadherin antibody……….80
Figure 22.    To analysis the activity of CX30.2/CX31.3 hemichannel functional via ATP release……………………….………...…81
Figure 23.    The effect of 18A-GA on extracellular ATP levels of CX30.2/CX31.3 mutants……………………………………….82

Chapter 4
Conclusions and Future work……………………………………………….………..83
Reference………………………………………………………………………….….86
Publication list………………………………………………………………………110

                                

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