Extracellular Cysteines Are Critical to Form Functional Cx46 Hemichannels

Ainoa Fernández-Olivares, Eduardo Durán-Jara, Daniel A. Verdugo, Mariana C. Fiori, Guillermo A. Altenberg, Jimmy Stehberg, Iván Alfaro, Juan Francisco Calderón, Mauricio A. Retamal

Research output: Contribution to journalArticlepeer-review

Abstract

Connexin (Cxs) hemichannels participate in several physiological and pathological pro-cesses, but the molecular mechanisms that control their gating remain elusive. We aimed at deter-mining the role of extracellular cysteines (Cys) in the gating and function of Cx46 hemichannels. We studied Cx46 and mutated all of its extracellular Cys to alanine (Ala) (one at a time) and studied the effects of the Cys mutations on Cx46 expression, localization, and hemichannel activity. Wild-type Cx46 and Cys mutants were expressed at comparable levels, with similar cellular localization. However, functional experiments showed that hemichannels formed by the Cys mutants did not open either in response to membrane depolarization or removal of extracellular divalent cations. Molecular-dynamics simulations showed that Cys mutants may show a possible alteration in the electrostatic potential of the hemichannel pore and an altered disposition of important residues that could contribute to the selectivity and voltage dependency in the hemichannels. Replacement of extracellular Cys resulted in “permanently closed hemichannels”, which is congruent with the inhibition of the Cx46 hemichannel by lipid peroxides, through the oxidation of extracellular Cys. These results point to the modification of extracellular Cys as potential targets for the treatment of Cx46-hemichannel associated pathologies, such as cataracts and cancer, and may shed light into the gating mechanisms of other Cx hemichannels.

Original languageEnglish
Article number7252
JournalInternational Journal of Molecular Sciences
Volume23
Issue number13
DOIs
Publication statusPublished - 1 Jul 2022

Keywords

  • channel permeability
  • connexins
  • extracellular loops
  • post-translational modification
  • redox sensing

ASJC Scopus subject areas

  • Catalysis
  • Molecular Biology
  • Spectroscopy
  • Computer Science Applications
  • Physical and Theoretical Chemistry
  • Organic Chemistry
  • Inorganic Chemistry

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