The p.Cys169Tyr variant of connexin 26 is not a polymorphism

Abstract

Mutations in the GJB2 gene, which encodes the gap junction protein connexin 26 (Cx26), are the primary cause of hereditary prelingual hearing impairment. Here, the p.Cys169Tyr missense mutation of Cx26 (Cx26C169Y), previously classified as a polymorphism, has been identified as causative of severe hearing loss in two Qatari families. We have analyzed the effect of this mutation using a combination of confocal immunofluorescence microscopy and molecular dynamics simulations. At the cellular level, our results show that the mutant protein fails to form junctional channels in HeLa transfectants despite being correctly targeted to the plasma membrane. At the molecular level, this effect can be accounted for by disruption of the disulfide bridge that Cys169 forms with Cys64 in the wild-type structure (Cx26WT). The lack of the disulfide bridge in the Cx26C169Y protein causes a spatial rearrangement of two important residues, Asn176 and Thr177. In the Cx26WT protein, these residues play a crucial role in the intra-molecular interactions that permit the formation of an intercellular channel by the head-to-head docking of two opposing hemichannels resident in the plasma membrane of adjacent cells. Our results elucidate the molecular pathogenesis of hereditary hearing loss due to the connexin mutation and facilitate the understanding of its role in both healthy and affected individuals.

Figure 1.

Pedigree of the families with indication of the phenotypes and the GJB2 genotypes. Pedigree showing three families with Cys169Tyr variants. For Family a, affected individuals are: II:1 (Cys169Tyr/ Cys169Tyr), II:2 (Cys169Tyr /Cys169Tyr) II:3 (Cys169Tyr/Cys169Tyr) and II:4; (Cys169Tyr /wt); Family b: the proband is II:1 (Cys169Tyr /wt); Family c: the proband is II:1 (C169Y/C169Y). Note: (Cys169Tyr /Cys169Tyr), homozygous; (Cys169Tyr /wt), heterozygous; wt, wild-type.

Figure 2.

Cx26 immunostaining performed in Hela DH transfectants overexpressing Cx26WT (top), and Cx26C169Y (bottom) proteins. Through focus confocal image sequence (z-stack) taken at 0.5 µm intervals of Hela DH cells expressing Cx26WT (A–D) and Cx26C169Y (F–I) proteins and their respective maximal projection rendering (E and J). Yellow arrow points to representative gap junction plaque, whereas red arrows indicate immunoreaction signals at the cell plasma membrane level, which are most likely due to unpaired connexons. Scale bar, 10 µm.

Figure 3.

Cartoon representation of Cx26WT (left) and Cx26C169Y (right) hemichannels. The six connexins composing the hemichannels are drawn in ribbon; the extracellular loops (EC1 and EC2) are shown in orange and red (respectively). The insets show details of a single connexin; residues 53–180, 60–174 and 64–169, which in the wild-type structure are linked by disulfide bonds, are drawn in licorice representation.

Figure 4.

Distance between residues 64 and 169 during molecular dynamics simulation. The graph on the left shows average distances between α carbons of the two residues (inset) for the Cx26WT hemichannel (blue) and the Cx26C169Y hemichannel (red) as a function of time. Frequency histograms for the distribution of distance values are presented in the right panel. The absence of the disulfide bridge in the mutant results in a broader distribution, which is also shifted toward larger values.

Figure 5.

The p.Cys169Tyr mutation of Cx26 perturbs also residues that are considered critical for hemichannel docking and formation of a full gap junction channel. The six left panels show the orientation of side chains of residues Asn176 and Thr177 in the six connexins of the most representative configurations of wild-type (blue) and mutant (red) hemichannels. Structural alteration in the extracellular loop due to the absence of one of the disulfide bridges alters the position of the side chains of these two critical residues in the mutant hemichannel, which are thought to be responsible of the formation a full gap junction channel. The six panels are ranked in order of increasing RMSD of α carbons of Asn176 and Thr177 from the crystal structure of (2). The RMSD values for these two residues are reported in the graph of the right (blue squares for Cx26WT, red diamonds for Cx26C169Y), together with those of all the seven residues that have been identified as responsible for the docking in (2): Asn54, Leu56, Gln57, Lys168, Asn176, Thr177, Asp179 (black triangles for Cx26WT, green circles for Cx26C169Y). The positions of residues Asn176 and Thr177 are the most affected by the mutation. The values reported for Cx26WT are weighted averages over the two most representative configurations, as explained in the Materials and Methods section.