Investigation of the molecular mechanisms underlying hearing loss mediated by connexin protein

• Main Authors: Tung-Cheng Li, 李東城
• Other Authors: 楊建洲
• Format: Others
• Language: en_US
• Published: 2014
• Online Access: http://ndltd.ncl.edu.tw/handle/61309457331907051791

博士 === 中山醫學大學 === 醫學研究所 === 102 === Hearing loss is the most common sensory disorder worldwide. The crucial role of gap junctions, which are composed of connexin (CX) protein, in auditory functions has been confirmed by numerous studies. In this study we are going to focus on the effect of mutations on the function of specific connexin genes [CX30.2/CX31.3 (GJC3), CX30.3 (GJB4) and CX31(GJB3)]. In a previous study, we have identified one novel missense mutation, p.W77S, in the GJC3 gene encoding connexin30.2/connexin31.3 (CX30.2/CX31.3) and a missense mutation, p.V174M, in the GJB3 gene encoding connexin 31 (Cx31), in the patient with nonsyndromic hearing loss. However, the functional change in the two missense mutations (CX30.2/CX31.3W77S and CX31V174M) remains unknown. In the study, our result indicated that the intracellular distribution of the p.W77S missense mutation proteins is different from that of CX30.2/CX31.3WT, which showed continuous staining along apposed cell membranes. Accumulation of the mutant protein in the endoplasmic reticulum (ER) of the HeLa cell was observed. Furthermore, co-expression of WT and p.W77S mutant proteins by a bi-directional tet-on expression system showed that the heteromeric connexon accumulated in the cytoplasm, thereby impairing the expression of the WT proteins in the cell membranes. In addition, we found that CX30.2/CX31.3W77S missense mutant proteins were degraded by lysosomes and proteosomes in the transfected HeLa cell. Based on these findings, we suggest that p.W77S mutant has a dominant negative effect on the formation and function of the gap junction. Additionally, we found that the p.V174M missense mutation resulted in the accumulation of the mutant protein in the lysosomes rather than in the cytoplasmic membrane in a fluorescent localization assay. This expression pattern is different from that of Cx31WT, which shows the typical punctuate pattern of a gap junction channel between the neighboring expression cells. Moreover, dye transfer experiments have also demonstrated that the CX31V174M mutant did not form functional gap junction channels, probably due to incorrect assemblies or altered properties of the CX31 channels. In addition, we found that CX31V174M-transfection can cause cell death by MTT assay. CX31V174M co-expressed with either CX31WT or CX26WT showed the impairment of the ability of CX26WT proteins to intracellular trafficking and targeting to the plasma membrane, but did not influence the trafficking of CX31WT. Based on these findings, we suggest that the CX31V174M mutant may have an effect on the formation and function of the gap junction and CX31V174M has a trans-dominant negative effect on the function of wild types CX26. Previously, we identified eleven patients with GJB4 gene variants in 253 unrelated Taiwanese patients with nonsyndromic hearing loss. In the study, we investigate the phenotype-genotype correlation in eleven deaf patients with variants of GJB4. Analytical results revealed that most probands had congenital hearing loss that is bilateral, stable and without associated dermatological manifestation or morphological changes of the inner ear. An audiometric profile including observed consistency, severe-to-profound and flat shapes dominant, may enable screening for variant of GJB4. On the basis of the results of investigation, we suggest that the GJB4 may be genetic risk factor for the development of nonsyndromic hearing loss in the Taiwanese, and our data can be applied to direct the clinical evaluation and effectively counsel families of children with GJB4.In conclusion, our study provides information for understanding the importance of genetic factors in nonsyndromic deafness of Taiwanese. These results provide a novel molecular explanation for the role CX plays in the development of hearing loss.