■为了探索两种常见变体的功能后果,p.V37I和c.299-300delAT在听力损失相关基因GJB2中的表达。
■在转染表达GJB2野生型质粒的HEK293T细胞中研究了连接蛋白26的表达和间隙连接通透性,p.V37I,或c.299-300delATCX26蛋白与荧光标签。进行了不同GJB2单倍型的功能分析,以全面评估离子和小分子偶联的改变。
■p.V37I蛋白定位于质膜,但未能有效转运细胞间碘化丙啶或Ca2+,表明生化和离子耦合的损害。GJB2p.V37I的存在似乎增加了细胞对H2O2处理的敏感性。相比之下,已知的变异c.299-300delAT蛋白不被转运到细胞膜上,不能形成间隙连接,而是局限于细胞质。在c.299-300delAT转染的细胞中,离子和生化偶联存在缺陷。
■p.V37I和c.299-300delATGJB2突变导致间隙连接介导的偶联缺陷。环境因素可能会影响GJB2p.V37I.的功能后果。这些结果可能会激发针对听力损失的GJB2突变的分子疗法的发展。
OBJECTIVE: The aim of this study was to explore the functional consequences of two common variants, p.V37I and c.299-300delAT, in the hearing loss-associated gene
GJB2.
METHODS: Connexin 26 expression and gap junctional permeability were studied in HEK 293T cells transfected with plasmids expressing
GJB2 wild-type, p.V37I, or c.299-300delAT CX26 proteins tagged with fluorescent markers. Functional analyses of various
GJB2 haplotypes were conducted to thoroughly evaluate alterations in ionic and small-molecule coupling.
RESULTS: The p.V37I protein was localized at the plasma membrane, but it failed to effectively transport intercellular propidium iodide or Ca2+ efficiently, indicating an impairment in both biochemical and ionic coupling. The presence of
GJB2 p.V37I seemed to increase the cells\' sensitivity to H2O2 treatment. In contrast, the known variant c.299-300delAT protein was not transported to the cell membrane and was unable to form gap junctions, remaining confined to the cytoplasm. Both ionic and biochemical coupling were defective in cells transfected with c.299-300delAT.
CONCLUSIONS: The p.V37I and c.299-300delAT GJB2 mutations resulted in deficient gap junction-mediated coupling. Additionally, environmental factors could influence the functional outcomes of the GJB2 p.V37I mutation. These findings could pave the way for the development of molecular therapies targeting
GJB2 mutations to treat hearing loss.