Deficient Gap Junction Coupling in Two Common Hearing Loss-Related Variants of GJB2
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Objectives. 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 plas- mids expressing GJB2 wild-type, p.V37I, or c.299-300delAT CX26 proteins tagged with fluorescent markers. Func- tional analyses of various GJB2 haplotypes were conducted to thoroughly evaluate alterations in ionic and small-mol- ecule coupling. Results. The p.V37I protein was localized at the plasma membrane, but it failed to effectively transport intercellular propid- ium 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 H O treatment. In contrast, the known variant c.299-300delAT22protein 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. Conclusion. The p.V37I and c.299-300delAT GJB2 mutations resulted in deficient gap junction-mediated coupling. Addi- tionally, 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.
Abstract
Objectives. 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 plas- mids expressing GJB2 wild-type, p.V37I, or c.299-300delAT CX26 proteins tagged with fluorescent markers. Func- tional analyses of various GJB2 haplotypes were conducted to thoroughly evaluate alterations in ionic and small-mol- ecule coupling. Results. The p.V37I protein was localized at the plasma membrane, but it failed to effectively transport intercellular propid- ium 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 H O treatment. In contrast, the known variant c.299-300delAT22protein 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. Conclusion. The p.V37I and c.299-300delAT GJB2 mutations resulted in deficient gap junction-mediated coupling. Addi- tionally, 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.
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