CX30.2/CX31.3基因之功能分析

Abstract

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.