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S. A. Molina, D. J. Takemoto; The Carboxy Terminal Tail of Connexin 46 Confers Resistance to Hypoxia in N2A Cells. Invest. Ophthalmol. Vis. Sci. 2010;51(13):4603.
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© ARVO (1962-2015); The Authors (2016-present)
Our lab has previously shown that rat Cx46 expression in N2A cells leads to a resistance to cellular death when exposed to hypoxia (1% O2). This effect is rCx46 specific when compared to rCx43. The purpose of this study is to investigate how the structure of rCx46 relates to its function as it confers resistance to hypoxia-induced cell death.
Mutants of rat Cx46 (Gja3) were constructed by PCR to contain amino acids 1-416 (WT), 1-235 (dCT) or 1-335 (tCT). The dCT and tCT mutants lacked a portion of the carboxy terminal tail region of the mature rCx46 protein, while the WT was full length rCx46. Mutants were individually cloned into the pEGFP-N3 vector to create rCx46-EGFP fusion proteins and were subsequently transfected into mouse neuroblastoma N2A cells using liposomes. Both transient and stable cell lines each expressing the versions of rCx46 were tested in a resazurin-based viability assay for survival in hypoxia (humidified 1% O2, 5% CO2, 94% N2). Additionally, WT, dCT, and tCT rCx46-EGFP were analyzed by fluorescence microscopy to observe cellular localization.
WT rCx46-EGFP conferred resistance to hypoxia-mediated cell death (~60% death), whereas dCT and tCT did not when compared to EGFP-only expressing N2A cells (~80% death) after 24 hours of culture in hypoxic conditions. The effect was more pronounced in stable cell lines where all cells affected by hypoxia were expressing rCx46-EGFP. WT rCx46-EGFP localized primarily to the perinuclear region and to some extent the cell membrane. The truncation mutants were also localized to the perinuclear region and not to the cell membrane.
The full length carboxy terminal tail of rCx46 is important in conferring N2A resistance to hypoxia-mediated cell death. A number of kinases phosphorylate the carboxy-terminus of rCx46 and may play an important role in the function of rCx46 beyond that of gap junctional coupling. Kinase activity on rCx46 is currently under study to understand how kinases affect the rCx46 protein during hypoxia. Additionally, cell membrane localized rCx46 is not necessary for N2A cellular resistance to hypoxia-induced cell death. This provides a possible intracellular role of rCx46 in prolonging cell survival during hypoxia in N2A cells.
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