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Gap junction channels formed by these connexins have been shown to have different permeability profiles, with Cx30 showing a strongly reduced preference for anionic tracers. The pore-forming segment of the first extracellular loop, E1, identified by computational studies of the Cx26 crystal structure to form a parahelix and a narrowed region of the pore, differs at a single residue at position 49. Cx26 contains an Ala and Cx30, a charged Glu at this position, and cysteine scanning in hemichannels identified this position to be pore-lining. To assess whether the Ala\/Glu difference affects permeability, we modeled and quantified Lucifer Yellow transfer between HeLa cell pairs expressing WT Cx26 and Cx30 and variants that reciprocally substituted Glu and Ala at position 49. Cx26(A49E) and Cx30(E49A) substitutions essentially reversed the Lucifer Yellow permeability profile when accounting for junctional conductance. Moreover, by using a calcein efflux assay in single cells, we observed a similar reduced anionic preference in undocked Cx30 hemichannels and a reversal with reciprocal Ala\/Glu substitutions. Thus, our data indicate that Cx26 and Cx30 gap junction channels and undocked hemichannels retain similar permeability characteristics and that a single residue difference in their E1 domains can largely account for their differential permeabilities to anionic tracers. The higher anionic permeability of Cx26 compared with Cx30 suggests that these connexins may serve distinct signaling functions in the cochlea, perhaps reflected in the vastly higher prevalence of Cx26 mutations in human deafness.<\/jats:p>","DOI":"10.1085\/jgp.202413600","type":"journal-article","created":{"date-parts":[[2024,8,29]],"date-time":"2024-08-29T02:20:34Z","timestamp":1724898034000},"update-policy":"https:\/\/doi.org\/10.1085\/jgp.crossmarkpolicy","source":"Crossref","is-referenced-by-count":6,"title":["An Ala\/Glu difference in E1 of Cx26 and Cx30 contributes to their differential anionic permeabilities"],"prefix":"10.1085","volume":"156","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-1057-5065","authenticated-orcid":true,"given":"Lina","family":"Kraujaliene","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/0069bkg23","id-type":"ROR","asserted-by":"publisher"}],"name":"Institute of Cardiology, Lithuanian University of Health Sciences 1 , Kaunas, Lithuania"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0007-6982-3653","authenticated-orcid":true,"given":"Tadas","family":"Kraujalis","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/0069bkg23","id-type":"ROR","asserted-by":"publisher"}],"name":"Institute of Cardiology, Lithuanian University of Health Sciences 1 , Kaunas, Lithuania"},{"id":[{"id":"https:\/\/ror.org\/01me6gb93","id-type":"ROR","asserted-by":"publisher"}],"name":"Kaunas University of Technology 2 Department of Applied Informatics, , Kaunas, Lithuania"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2132-519X","authenticated-orcid":true,"given":"Mindaugas","family":"Snipas","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/0069bkg23","id-type":"ROR","asserted-by":"publisher"}],"name":"Institute of Cardiology, Lithuanian University of Health Sciences 1 , Kaunas, Lithuania"},{"id":[{"id":"https:\/\/ror.org\/01me6gb93","id-type":"ROR","asserted-by":"publisher"}],"name":"Kaunas University of Technology 3 Department of Mathematical Modelling, , Kaunas, Lithuania"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6647-1102","authenticated-orcid":true,"given":"Vytas K.","family":"Verselis","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/05cf8a891","id-type":"ROR","asserted-by":"publisher"}],"name":"Albert Einstein College of Medicine 4 Dominick P. 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