A pore locus in the E1 domain differentially regulates Cx26 and Cx30 hemichannel function.

Connexins (Cxs) function as gap junction (GJ) channels and hemichannels that mediate intercellular and transmembrane signaling, respectively. Here, we investigated the proximal segment of the first extracellular loop, E1, of two closely related Cxs, Cx26 and Cx30, that share widespread expression in the cochlea. Computational studies of Cx26 proposed that this segment of E1 contains a parahelix and functions in gating. The sequence of the parahelix is identical between Cx26 and Cx30 except for an Ala/Glu difference at position 49. We show through cysteine-scanning and mutational analyses that position 49 is pore-lining and interacts with the adjacent Asp50 residue to impact hemichannel functionality. When both positions 49 and 50 are charged, as occurs naturally in Cx30, the hemichannel function is dampened. Co-expression of Cx30 with Cx26(D50N), the most common mutation associated with keratitis-ichthyosis-deafness syndrome, results in robust hemichannel currents indicating that position 49-50 interactions are relevant in heteromerically assembled hemichannels. Cysteine substitution at position 49 in either Cx26 or Cx30 results in tonic inhibition of hemichannels, both through disulfide formation and high-affinity metal coordination, suggestive of a flexible region of the pore that can narrow substantially. These effects are absent in GJ channels, which exhibit wild-type functionality. Examination of postnatal cochlear explants suggests that Cx30 expression is associated with reduced propagation of Ca2+ waves. Overall, these data identify a pore locus in E1 of Cx26 and Cx30 that impacts hemichannel functionality and provide new considerations for understanding the roles of these connexins in cochlear function.

GJB2 and GJB6 Genetic Variant Curation in an Argentinean Non-Syndromic Hearing-Impaired Cohort.

Genetic variants in GJB2 and GJB6 genes are the most frequent causes of hereditary hearing loss among several deaf populations worldwide. Molecular diagnosis enables proper genetic counseling and medical prognosis to patients. In this study, we present an update of testing results in a cohort of Argentinean non-syndromic hearing-impaired individuals. A total of 48 different sequence variants were detected in genomic DNA from patients referred to our laboratory. They were manually curated and classified based on the American College of Medical Genetics and Genomics/Association for Molecular Pathology ACMG/AMP standards and hearing-loss-gene-specific criteria of the ClinGen Hearing Loss Expert Panel. More than 50% of sequence variants were reclassified from their previous categorization in ClinVar. These results provide an accurately interpreted set of variants to be taken into account by clinicians and the scientific community, and hence, aid the precise genetic counseling to patients.

Polydisperse molecular architecture of connexin 26/30 heteromeric hemichannels revealed by atomic force microscopy imaging.

Connexin (Cx) protein forms hemichannels and gap junctional channels, which play diverse and profound roles in human physiology and diseases. Gap junctions are arrays of intercellular channels formed by the docking of two hemichannels from adjacent cells. Each hexameric hemichannel contains the same or different Cx isoform. Although homomeric Cxs forms have been largely described functionally and structurally, the stoichiometry and arrangement of heteromeric Cx channels remain unknown. The latter, however, are widely expressed in human tissues and variation might have important implications on channel function. Investigating properties of heteromeric Cx channels is challenging considering the high number of potential subunit arrangements and stoichiometries, even when only combining two Cx isoforms. To tackle this problem, we engineered an HA tag onto Cx26 or Cx30 subunits and imaged hemichannels that were liganded by Fab-epitope antibody fragments via atomic force microscopy. For Cx26-HA/Cx30 or Cx30-HA/Cx26 heteromeric channels, the Fab-HA binding distribution was binomial with a maximum of three Fab-HA bound. Furthermore, imaged Cx26/Cx30-HA triple liganded by Fab-HA showed multiple arrangements that can be derived from the law of total probabilities. Atomic force microscopy imaging of ringlike structures of Cx26/Cx30-HA hemichannels confirmed these findings and also detected a polydisperse distribution of stoichiometries. Our results indicate a dominant subunit stoichiometry of 3Cx26:3Cx30 with the most abundant subunit arrangement of Cx26-Cx26-Cx30-Cx26-Cx30-Cx30. To our knowledge, this is the first time that the molecular architecture of heteromeric Cx channels has been revealed, thus providing the basis to explore the functional effect of these channels in biology.

Exome Sequencing Identifies a Novel Nonsense Mutation of MYO6 as the Cause of Deafness in a Brazilian Family.

We investigated 313 unrelated subjects who presented with hearing loss to identify the novel genetic causes of this condition in Brazil. Causative GJB2/GJB6 mutations were found in 12.7% of the patients. Among the familial cases (100/313), four were selected for exome sequencing. In one case, two novel heterozygous variants were found and were predicted to be pathogenic based on bioinformatics tools, that is, p.Ser906* (MYO6) and p.Arg42Cys (GJB3). We confirmed that this nonsense MYO6 mutation segregated with deafness in this family. Only the proband and her unaffected mother exhibited the GJB3 mutation, which is in the same amino acid of a known Erythrokeratodermia variabilis mutation. None of the patients exhibited this skin disease, but the proband exhibited a more severe hearing loss. Hence, the GJB3 mutation was considered to be a variant of uncertain significance. In conclusion, we described a novel nonsense MYO6 mutation that was responsible for the hearing loss in a Brazilian family. This mutation resides in the neck domain of myosin-VI after the motor domain. Thus, our data give further support for genotype-phenotype correlations, which state that when the motor domain of the protein is functioning, the hearing loss is milder and has a later onset. The three remaining families without mutations in the known genes suggest that there are still deafness genes to be revealed.