OpenAIRE Collection / OpenAIRE Koleksiyonu
Permanent URI for this collectionhttps://hdl.handle.net/11147/17
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Article Alteration of Protein Localization and Intracellular Calcium Content Due To Connexin26 D50a and A88v Mutations(Türk Biyokimya Derneği, 2017) Aypek, Hande; Meşe, GülistanIntroduction: Connexins (Cx) play essential roles in cellular homeostasis by forming gap junctions and non-junctional hemichannels. In vitro characterization of Cx26 mutations causing keratitis-ichthyosis-deafness (KID) syndrome, were shown to form leaky hemichannels. The molecular/ cellular mechanisms affected by aberrant hemichannels have recently been elucidated. Here, we further wanted to characterize Cx26 KID syndrome mutations, D50A and A88V, which were shown to form aberrant hemichannels and remained unaddressed in the literature. Methods: Neurobiotin uptake assay in HeLa and N2A cells transfected with Cx26-WT, D50A or A88V verified the presence of aberrant hemichannels and immunofluorescent staining with fluorescent microscopy determined cellular localization of Cx26. Finally, intracellular calcium content was examined by using calcium indicator, Fluo-3AM, and flow cytometer. Results: Cx26-D50A and A88V mutations prevented the formation of gap junction plaques at cell-cell appositions and mutant proteins were observed to localize to the Golgi apparatus. Further, comparison of intracellular calcium content showed an increase in calcium amount in cells containing Cx26-D50A and A88V relative to Cx26-WT. Conclusion: Retention of Cx26 in the Golgi apparatus and alteration in the intracellular calcium content due to KID syndrome mutations may influence various cellular processes that might contribute to development of epidermal phenotypes.Article Citation - WoS: 14Citation - Scopus: 16Altered Cellular Localization and Hemichannel Activities of Kid Syndrome Associated Connexin26 I30n and D50y Mutations(BioMed Central Ltd., 2016) Aypek, Hande; Meşe Özçivici, Gülistan; Bay, Veysel; Meşe Özçivici, Gülistan; Meşe, GülistanBackground: Gap junctions facilitate exchange of small molecules between adjacent cells, serving a crucial function for the maintenance of cellular homeostasis. Mutations in connexins, the basic unit of gap junctions, are associated with several human hereditary disorders. For example, mutations in connexin26 (Cx26) cause both non-syndromic deafness and syndromic deafness associated with skin abnormalities such as keratitis-ichthyosis-deafness (KID) syndrome. These mutations can alter the formation and function of gap junction channels through different mechanisms, and in turn interfere with various cellular processes leading to distinct disorders. The KID associated Cx26 mutations were mostly shown to result in elevated hemichannel activities. However, the effects of these aberrant hemichannels on cellular processes are recently being deciphered. Here, we assessed the effect of two Cx26 mutations associated with KID syndrome, Cx26I30N and D50Y, on protein biosynthesis and channel function in N2A and HeLa cells. Results: Immunostaining experiments showed that Cx26I30N and D50Y failed to form gap junction plaques at cell-cell contact sites. Further, these mutations resulted in the retention of Cx26 protein in the Golgi apparatus. Examination of hemichannel function by fluorescent dye uptake assays revealed that cells with Cx26I30N and D50Y mutations had increased dye uptake compared to Cx26WT (wild-type) containing cells, indicating abnormal hemichannel activities. Cells with mutant proteins had elevated intracellular calcium levels compared to Cx26WT transfected cells, which were abolished by a hemichannel blocker, carbenoxolone (CBX), as measured by Fluo-3 AM loading and flow cytometry. Conclusions: Here, we demonstrated that Cx26I30N and D50Y mutations resulted in the formation of aberrant hemichannels that might result in elevated intracellular calcium levels, a process which may contribute to the hyperproliferative epidermal phenotypes of KID syndrome.