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.

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.