Clin Exp Otorhinolaryngol.  2024 Aug;17(3):198-205. 10.21053/ceo.2023.00078.

Deficient Gap Junction Coupling in Two Common Hearing Loss-Related Variants of GJB2

• Chen K
• Jiang H

• KMID: 2558836
• DOI: http://doi.org/10.21053/ceo.2023.00078

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 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.
Conclusion
. 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.

Keyword

Connexin 26; Genetic Susceptibility; Gap Junction; GJB2; Sensorineural Hearing Loss

Figure

• 

  Fig. 1. (A-F) Immunofluorescence staining results after transfection with various CX26 vectors. Compared with the massive plaques in HEK 293T cells expressing CX26-c.299-300delAT (white arrows in E and F) or empty vector (D), intercellular gap junctions (yellow arrows in A-C) were observed. Scale bar=10 μm. (G) The percentages of gap junctions formed in three haplotype are shown. DAPI, 4´,6-diamidino-2´-phenylindole; GFP, green fluorescent protein; NS, not significant.

• 

  Fig. 2. Propidium iodide (PI) permeability by gap junctions formed by the four CX26 haplotypes in HEK 293T cells. Asterisks indicate cells that were injected with PI. (A-D) Representative images of cells expressing CX26-WT (A) or CX26-WT/p.V37I (C), showing the detection of PI dye in neighboring cells with gap junctions (green), in contrast to leaky PI transfer capacity in cells expressing CX26-p.V37I (B) or c.299-300delAT (D). Scale bar=10 μm. (E) Cell count analysis supporting the severe alteration in PI loading in CX26-p.V37I cells. NS, not significant. **P<0.01.

• 

  Fig. 3. Hemichannel dye loading assay of HEK 293T cells expressing the four connexin 26 (CX26) haplotypes. (A-D) Representative images of cells loaded with the membrane-impermeable fluorescent dye propidium iodide (PI). Scale bar=10 μm. (E) Quantification of the PI loading rate, showing a significant reduction in cells expressing CX26-p.V37I compared with that in cells expressing CX26-WT. NS, not significant. **P<0.01.

• 

  Fig. 4. (A, B) Ionic permeability of gap junctions (GJs) in HEK 293T cells were functional in cells expressing the CX26-WT and CX26-WT/p.V37I haplotypes. (C, D) Impaired permeability of ionic GJs was detected in CX26-p.V37I and CX26-c.299-300delAT. Scale bar= 10 μm. (E) The Ca2+ transfer rates are shown. **P<0.01.

• 

  Fig. 5. (A) Representative results showing the morphological changes of HEK 293T cells with three haplotypes after H2O2 incubation. Scale bar=50 μm. (B) The tendency for apoptosis in HEK 293T cells expressing CX26-WT and/or CX26-p.V37I, treated with 20 μM to 80 μM H2O2. (C) Apoptosis rate. (D) Dynamic changes in biochemical hemichannel function in cells carrying three vectors. SSC-H, side scatter height; FITC, fluorescein isothiocyanate; NS, not significant. *P<0.05 when comparing the values in CX26-p.V37I with the values in CX26-WT or CX26-WT/p.V37I haplotypes.

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