Critical functional role of arginine 75 in connexin 26 gap‐junction channels and hemichannels

Gap‐junction channels (GJC), formed by two gap‐junction hemichannels (GJH) docked head‐to‐head, mediate cell‐to‐cell communication. Each GJH is a connexin (Cx) hexamer. Cx26 is expressed in several cell types of the cochlea and expression of the Cx26‐R75W mutant causes dominant deafness. Our current understanding is insufficient to explain how Cx26 mutations cause deafness. Here, we studied the effects of Cx26‐R75 substitutions on the function of GJC and GJH in paired and single Xenopus oocytes, respectively. We confirmed that Cx26‐R75W does not form functional GJC in paired oocytes, although it forms functional GJH. The mutant GJH have the same single‐GJH conductance as wild‐type (WT) Cx26 GJH, and are also blocked by extracellular Ca 2+ and known GJC blockers. However, they have a reduced open probability at negative membrane voltages. To ascertain the properties of the side chain at position 75 required to form functional GJC, we studied additional mutants. Cx26‐R75Y and Cx26‐R75F behave like Cx26‐R75W (they showed GJH, but no GJC currents). In contrast, Cx26‐R75A, Cx26‐R75N, Cx26‐R75D and Cx26‐R75K did not form GJH or GJC, although they were expressed in the plasma membrane. Expression of Cx26‐R75W, Cx26‐R75Y or Cx26‐R75A had a dominant negative effect on GJC formed by WT Cx26, independently on whether they form functional GJH or not. Our results indicate that R at position 75 is essential for Cx26 function, i.e., it cannot be replaced by other residues with preservation of GJC function. An aromatic side chain seems sufficient to form functional GJH, although their properties are different from those of WT Cx26 GJH. Supported in part by Sealy Endowment Fund for Biomedical Research and the National Institutes of Health.